Method of improving the quality of stored potatoes

ABSTRACT

The invention relates to a method of improving the quality of potatoes stored at reduced temperatures and a method of prolonging dormancy of stored potato tubers, by increasing the level of ADPglucose pyrophosphorylase enzyme activity within the potato tuber during storage at ambient or reduced temperatures. Novel DNA molecules, plant cells, and potato plants are provided which contain the gene for the ADPglucose pyrophosphorylase enzyme.

This application is a continuation-in-part of Ser. No. 070,155, filedMay 28, 1993, now abandoned.

BACKGROUND OF THE INVENTION

Long term storage properties of potato represents a major determinant oftuber quality. Dormancy periods (the time period after harvesting andbefore sprouting) are crucial to maintaining quality potatoes.Commercially, potatoes may be held for long periods before processing(up to 10 months or longer), and at temperatures typically between2°-10° C. Cold storage (2°-6° C.) versus storage at 7°-12° C. providesthe best long term conditions by reducing respiration, water loss,microbial infection, and the need for chemical sprout inhibitors(Burton, 1989). However, low temperatures lead to cold-inducedsweetening, and the resultant high sugar levels contribute to anunacceptable brown color in the fried product (Coffin et al., 1987,Weaver et al., 1978). The sugars that accumulate are predominantlyglucose, fructose, and sucrose, and it is mainly the reducing sugars(primarily glucose and fructose) which react with free amino groups uponheating during the various cooking processes, including frying, via theMaillard reaction, and result in the formation of brown pigment (Burton,1989, Shallenberger et al., 1959). Sucrose, on the other hand, producesa black coloration on frying due to its susceptibility to undergocarmelization as well as charring. Levels of reducing sugars above 0.2%fresh weight are sufficient to cause brown pigment formation and thusmerit rejection for certain types of processing. A potato processor canreduce the levels of sugars by a costly and time consuming blanchingprocess if the levels of sugars are not significantly higher than the0.2% limit. Potatoes can be reconditioned at higher temperatures (18°C.) to lower sugar content, but often sugar levels will not sufficientlydecrease before the onset of sprouting at these temperatures, requiringthe use of chemical sprout inhibitors (Ryall and Lipton, 1979,Hardenburg et al., 1986). However, reconditioning increases the storagefacility requirements and consequently affects the final cost of theproduct. Furthermore, it has been shown that reconditioning is noteffective after longer storage periods (Coffin et al., 1987). Given thenegative environmental and health perceptions associated with excessivechemical use, and the fact that current sprout inhibitors may soon bebanned, a need exists for potato varieties which can withstand long termcold storage without the use of chemicals, without accumulation ofreducing sugars, and with greater retention of starch levels.

After longer storage periods, sprouting of potato tubers becomes aproblem. Excess sprouting reduces the market value and can causeincreased levels of alkaloids in the tuber.

Through the process of genetic engineering potato tubers which containsignificantly higher levels of starch have been obtained. See WO91/19806 (Kishore), also U.S. Ser. No. 07/709,663, filed Jun. 7, 1991,now abandoned, hereby incorporated by reference. In these tubers a geneis expressed which encodes ADPglucose pyrophosphorylase (ADPGPP), whichcatalyzes a key step in starch and glycogen biosynthesis. The preferredgene is from E. coli and the resulting enzyme is a poorly regulated,highly active variant. When a mutant of this gene, glgC16, is expressedin a tuber-specific manner, for example from a class I patatin promoter,starch levels are higher than those of nontransgenic control tubers atthe time of harvest.

Carbohydrate metabolism is a complex process in plant cells.Manipulation of a number of different enzymatic processes potentiallymay effect the accumulation of reducing sugars during cold storage. Forexample, sugars may be used to resynthesize starch, and thus effectreduction in the pool of free sugar. Other methods may also serve toenhance the cold storage properties of potato through reduction of sugarcontent, including the inhibition of starch hydrolysis, removal ofsugars through glycolysis, or conversion of sugars into other forthswhich would not participate in the Maillard reaction. The challenge inthese methods would be to identify an activity with which to effect thedesired result, achieve function at low temperatures, and still retainthe product qualities desired by potato growers, processors, andconsumers.

It has been suggested that phosphofructokinase (PFK) plays an importantrole in the cold-induced sweetening process (Kruger and Hammond, 1988,ap Rees et al., 1988, Dixon et al., 1981, Claassen et al., 1991). apRees et al. (1988) suggested that cold treatment had a disproportionateeffect on different pathways in carbohydrate metabolism in thatglycolysis was more severely reduced due to the cold-lability of PFK.The reduction in PFK activity would then lead to an increasedavailability of hexose-phosphates for sucrose production. Additionalsupport for this view comes from the observation of a new breeding cloneof potato which contains a PFK which is not cold labile and that doesnot accumulate significant amounts of sugar in the cold.

It was recently disclosed in European Patent Application 0 438 904 thatincreasing PFK activity reduces sugar accumulation during storage byremoving hexoses through glycolysis and further metabolism. A PFK enzymefrom E. coli was expressed in potato tubers and the report claimed toincrease PFK activity and to reduce sucrose content in tubers assayed atharvest. However, it has been shown that pyrophosphate:Fructose6-phosphate phosphotransferase (PFP) remains active at low temperatures(Claassen et al., 1991). PFP activity can supply fructose 6-phosphatefor glycolysis just as PFK can since the two enzymes catalyze the samereaction. Therefore the efficacy of this approach in improving the coldstorage quality of potato tubers remains in doubt. Furthermore, theremoval of sugars through glycolysis and further metabolism would not bea preferred method of enhancing storage properties of potato tubersbecause of the resultant loss of valuable dry matter content throughrespiration. Resynthesis of the sugars into starch or slowing thebreakdown of starch would be preferred because dry matter would beretained.

It is an object of this invention to provide a method for reducing thelevel of sugars within potato tubers and to provide improved quality ofstored potatoes. It is a further object of this invention to providepotatoes having an improved rate and degree of reconditioning afterstorage at reduced temperatures. It is a still further object of thisinvention to provide a method of extending dormancy of potatoes storedat ambient temperatures or at reduced temperatures.

SUMMARY OF THE INVENTION

The present invention provides a method of improving the quality ofpotatoes stored at low temperatures comprising providing an increasedlevel of ADPglucose pyrophosphorylase (ADPGPP) enzyme activity withinthe potato tuber during storage at reduced temperatures. Also providedis a method of reducing the level of sugars within potato tubers storedat reduced temperatures by increasing the ADPGPP enzyme activity duringcold storage. Further provided is a method of prolonging dormancy ofstored potatoes comprising increasing the ADPGPP enzyme activity duringstorage.

This method is preferably accomplished by:

(a) inserting into the genome of a potato plant cell a recombinant,double-stranded DNA molecule comprising

(i) a promoter which functions in plants to cause the production of anRNA sequence in target plant tissues,

(ii) a structural DNA sequence that causes the production of an RNAsequence which encodes a fusion polypeptide comprising an amino-terminalplastid transit peptide and an ADPglucose pyrophosphorylase enzyme,

(iii) a 3' nontranslated DNA sequence which functions in plant cells tocause transcriptional termination and the addition of polyadenylatednucleotides to the 3' end of the RNA sequence;

(b) obtaining transformed plant cells; and

(c) regenerating from the transformed plant cells geneticallytransformed potato plants which have improved cold storage properties.

Novel recombinant DNA molecules, plant cells, and regenerated potatoplants are provided wherein the promoter of (a)(i) is a cold-induciblepromoter, such as from potato or Arabidopsis. These regenerated potatoplants are useful in all of the methods of the present invention.

A preferred ADPglucose pyrophosphorylase (ADPGPP) enzyme is that from E.coli, known as glgC, which gene sequence is shown below as SEQ ID NO:1and which amino acid sequence is shown as SEQ ID NO:2. A more preferredADPGPP enzyme is the mutant ADPGPP, glgC16, which gene sequence is shownbelow as SEQ ID NO:3 and which amino acid sequence is shown below as SEQID NO:4. This mutant has been found to have a higher affinity tosubstrates in the absence of the activator, fructose 1,6-bisphosphate(FBP), and to reach half-maximal activation with a decreasedconcentration of FBP.

As used herein, the term "improving the quality of stored potatoes," orvariants thereof, shall mean providing potatoes which after storage havereduced levels of sugars, little or no loss of starch, reduced incidenceof sprouting, and/or an enhanced rate or degree of reconditioning.

As used herein, the term "cold storage" or "storage at reducedtemperature," or variants thereof, shall mean holding at temperaturesless than or equal to 15° C., which may be caused by refrigeration orambient temperatures.

As used herein, the term "cold-inducible promoter" shall mean a sequenceof DNA bases that initiates the transcription of mRNA using one of theDNA strands as a template to make a corresponding complimentary strandof RNA when the temperature is equal to or less than 15° C.

As used herein, the term "prolonging dormancy" or variants thereof shallmean delaying onset of respiration and sprouting of tubers.

As used herein, the term "glgC16 potatoes," "glgC16 tubers," "glgC16lines," or variants thereof, shall mean potato lines or tubers therefromwhich have been transformed with a fusion of a plastid terminal transitpeptide, preferably CTP, described below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a plasmid map for plant transformation vector pMON17316.

FIG. 2 shows a plasmid map for plant transformation vector pMON17279.

DETAILED DESCRIPTION OF THE INVENTION

Starch phosphorylase and amylolytic enzymes are responsible for starchdegradation during cold storage and result in the formation of glucose1-phosphate and/or glucose from starch. Glucose may be convened toglucose 1-phosphate and serve as a substrate for the ADPGPP enzyme andthus for starch biosynthesis in the tubers expressing this enzyme.Glucose 1-phosphate may also be formed from the products of degradationof sucrose via invertase or sucrose synthase. Reducing sugars primarilyaccumulate during storage rather than sucrose due to the action ofinvertase (Pressey, 1966). The glucose and fructose released frominvertase activity can also serve as precursors of substrates for starchbiosynthesis.

The expression of ADPGPP is an effective means of countering the effectsof cold-induced sweetening. It is hypothesized that by maintainingstarch biosynthesis during cold storage, the continuous demand on thehexose pool is such that sugar accumulation is reduced and thus thetuber remains suitable for processing. However, other mechanisms mayalso be responsible for this effect of ADPGPP. In addition, prolongingthe dormancy of potatoes stored at any temperature may also beaccomplished by keeping the sugar level low and delaying onset ofrespiration and thus sprouting.

In order to accomplish the foregoing, a gene for expression of ADPGPP isincorporated within the genome of potato plants. This gene may becombined with other genes (in sense or antisense orientation) forregulation of starch and/or sugar metabolism/catabolism in potatoes, forexample, phosphofrutcokinases (EP 0 438 904); α- and β-amylases; sucrosephosphate synthases; hexokinases; starch phosphorylases; debranchingenzymes; or phosphoglucomutases. These additional genes may be from aplant, microorganism, or animal source.

Alternatively, increased levels of ADPGPP in stored tubers may beachieved by mutagenizing potato clones and thus increasing ADPGPP enzymeactivity levels. Such tubers could be selected based on display ofincreased specific activity, increased V_(max), reduced inhibition bythe negative effector (P_(i)), or reduced dependence upon activator(3-PGA) for maximal activity.

The expression of a plant gene which exists in double-stranded DNA forminvolves transcription of messenger RNA (mRNA) from one strand of theDNA by RNA polymerase enzyme, and the subsequent processing of the mRNAprimary transcript inside the nucleus. This processing involves a 3'nontranslated region which adds polyadenylate nucleotides to the 3' endof the RNA.

Transcription of DNA into mRNA is regulated by a region of DNA usuallyreferred to as the "promoter." The promoter region contains a sequenceof bases that signals RNA polymerase to associate with the DNA, and toinitiate the transcription of mRNA using one of the DNA strands as atemplate to make a corresponding complimentary strand of RNA.

A number of promoters which are active in plant cells have beendescribed in the literature. These include the nopaline synthase (NOS)and octopine synthase (OCS) promoters (which are carried ontumor-inducing plasmids of Agrobacterium tumefaciens), the caulimoviruspromoters such as the cauliflower mosaic virus (CaMV) 19S and 35S andthe figwort mosaic virus 35S-promoters, the light-inducible promoterfrom the small subunit of ribulose-1,5-bis-phosphate carboxylase(ssRUBISCO, a very abundant plant polypeptide), and the chlorophyll a/bbinding protein gene promoter, etc. All of these promoters have beenused to create various types of DNA constructs which have been expressedin plants; see, e.g., PCT publication WO 84/02913.

The class I patatin promoters used in Examples 1 and 2 below, have beenshown to be both highly active and tuber-specific (Bevan et al., 1986;Jefferson et al., 1990). A number of other genes with tuber-specific orenhanced expression are known, including the potato tuber ADPGPP genes,large and small subunits (Muller et al., 1990), sucrose synthase(Salanoubat and Belliard, 1987, 1989), the major tuber proteinsincluding the 22 kd protein complexes and proteinase inhibitors(Hannapel, 1990), the granule bound starch synthase gene (GBSS) (Rohdeet al., 1990), and the other class I and II patatins (Rocha-Sosa et al.,1989; Mignery et al., 1988). Other promoters which are contemplated tobe useful in this invention include those that show enhanced or specificexpression in potato tubers, that are promoters normally associated withthe expression of starch biosynthetic or modification enzyme genes, orthat show different patterns of expression within the potato tuber, withcortex-or pith- or periderm-enhanced expression, for example, or areexpressed at different times during tuber development. Examples of thesepromoters include those for the genes for the granule-bound and otherstarch synthases, the branching enzymes (Kossmann et al., 1991; Bleunow,A. and Johansson, G., 1991; WO 92/14827; WO 92/11375),disproportionating enzyme (Takaha et al., 1993), debranching enzymes,amylases, starch phosphorylases (Nakano et al., 1989; Mori et al.,1991), pectin esterases (Ebbelaar, et al., 1993), the 40 kDglycoprotein; ubiquitin, aspartic proteinase inhibitor (Stukerlj et al.,1990), the carboxypeptidase inhibitor, tuber polyphenol oxidases (Shaharet al., 1992; GenBank® Accession Numbers M95196 and M95197), putativetrypsin inhibitor and other tuber cDNAs (Stiekema et al., 1988), and forβ-amylase and sporarnins (from Ipomoea batatas; Yoshida et al., 1992;Ohta et al., 1991).

Expression of bacterial ADPGPP from various potato promoters has beenshown by Kishore in PCT Application WO 91/19806 to result in an increasein starch content in potato tubers.

It is not a requirement of the present method to start with a tuber withhigh starch content to achieve low reducing sugar accumulation duringcold storage. The glgC16 gene can be expressed from a cold-inducedpromoter in potato so that the GlgC16 enzyme is only present duringstorage conditions. The presence of this enzyme would then maintainstarch biosynthesis during storage and thus prevent the accumulation ofsugars.

Examples of cold-inducible promoters, including plant promoters arenumerous (Yamaguchi-Shinozaki et al., 1993; Qoronfleh et al., 1992;Miner et al., 1992; Houde et al., 1992; White et al., 1992; Huang etal., 1987; Murata et al., 1992; Gilmour et al., 1992, Hajela et al.,1990; and Kurkela et al., 1990). Isolation of cold-induced proteins inpotato tubers has been demonstrated (van Berkel et al., 1991, van Berkelet al., 1994). The promoters driving cold-induced expression of theseproteins can be isolated by methods available to those skilled in theart. One method involves production of a cDNA library from cold stressedtubers and subsequent identification of cold-specific clones bydifferential hybridization with a non-stressed library. This process canbe made more efficient by using subtraction libraries wherein clonesexpressed in a non-cold-specific manner are removed from the libraryduring construction. The determination of the nucleotide sequences ofcDNA's derived from these regulated transcripts will also facilitate theisolation of the corresponding promoter regions. The sequences of suchcDNAs are know for a number of the potato tuber cold regulatedtranscripts (van Berkel et al., 1994). The promoter fragment could thenbe identified from a genomic clone using cDNA probes identified ascold-specific. Such cold regulated promoters have been identified andsequenced (Yamaguchi-Shinozaki and Shinozaki, 1994, and Baker, 1994).The promoter fragment can be used to direct expression of the E. coliglgC16 gene in a cold-induced manner. Additionally, one of several otherADPGPP enzymes could be expressed from this promoter to affect sugarconcentration in cold stored potato tubers and thus improve the qualityof the tubers. Hybrid promoters or fusions of regulatory elements ofdifferent promoters may also be employed to increase the expressionlevel of a cold regulated promoter or to make such expression morespecific to the desired plant organ. Cold regulated genes have beendescribed in which the expression is preferential in different tissues(Zhu et at., 1993) or in which the genes are regulated more specificallyby cold than by other stress effects (Wilhelm and Thomashow, 1993;Nordin et al., 1993). In addition, specific defined sequences, of sizesfrom 9 base pairs to a few hundred base pairs in length, have been shownto control the reponsiveness of the promoters to different cold and toother stress effects such as abscisic acid levels and to drought stress(Yamaguchi-Shinozaki and Shinozaki, 1994 ). Promoters that expresspreferentially in tubers are know and the regions of these promotersthat are necessary for this preferential expression have also beendetermined (Jefferson et al., 1990; Liu et al., 1990). These data enablethe construction of fusions between the small cold responsive elementfrom promoters such as those from cor78, cor 15a, or cor15b, forexample, and a patatin promoter. Fusions are made to the -500 to -2000bp region of the patatin promoter. Current molecular genetic techniques,including Polymerase Chain Reaction and site-directed mutagenesis, andthe facility of oligonucleotide synthesis make these fusions possible.

The amino-terminal plastid transit peptide used with the ADPGPP gene isneeded to transport the enzyme to the plastid where starch synthesisoccurs. Alternatively, the transit peptide could be omitted and the genecould be inserted into the DNA present in the plastid. Chloroplasttransformation may be accomplished using the methods described by Svabet .al., 1990.

Production of Altered ADPglucose Pyrophosphorylase Genes by Mutagenesis

Those skilled in the art will recognize that while not absolutelyrequired, enhanced results are to be obtained by using ADPglucosepyrophosphorylase genes which are subject to reduced allostericregulation ("deregulated") and more preferably not subject tosignificant levels of allosteric regulation ("unregulated") whilemaintaining adequate catalytic activity. The structural coding sequencefor a bacterial or plant ADPglucose pyrophosphorylase enzyme can bemutagenized in E. coli or another suitable host and screened forincreased glycogen production as described for the glgC16 gene or E.coli. It should be realized that use of a gene encoding an ADPglucosepyrophosphorylase enzyme which is only subject to modulators(activators/inhibitors) which are present in the selected plant atlevels which do not significantly inhibit the catalytic activity willnot require enzyme (gene) modification. These "unregulated" or"deregulated" ADPglucose pyrophosphorylase genes can then be insertedinto plants as described herein to obtain transgenic plants havingincreased starch content.

For example, any ADPglucose pyrophosphorylase gene can be cloned intothe E. coli B strain AC70R1-504 (Leung, 1986). This strain has adefective ADPglucose pyrophosphorylase gene, and is depressed five- toseven-fold for the other glycogen biosynthetic enzymes. The ADPglucosepyrophosphorylase gene/cDNA's can be put on a plasmid behind the E. coliglgC promoter or any other bacterial promoter. This construct can thenbe subjected to either site-directed or random mutagenesis. Aftermutagenesis, the cells would be plated on rich medium with 1% glucose.After the colonies have developed, the plates would be flooded withiodine solution (0.2 w/v % I₂, 0.4 w/v % KI in H₂ O, Creuzet-Sigal,1972). By comparison with an identical plate containing non-mutated E.coli, colonies that are producing more glycogen can be detected by theirdarker staining.

Since the mutagenesis procedure could have created promoter mutations,any putative ADPglucose pyrophosphorylase mutant from the first roundscreening will have to have the ADPglucose pyrophosphorylase generecloned into non-mutated vector and the resulting plasmid will bescreened in the same manner. The mutants that make it though both roundsof screening will then have their ADPglucose pyrophosphorylaseactivities assayed with and without the activators and inhibitors. Bycomparing the mutated ADPglucose pyrophosphorylase's responses toactivators and inhibitors to the non-mutated enzymes, the new mutant canbe characterized.

The report by Plaxton and Preiss in 1987 demonstrates that the maizeendosperm ADPglucose pyrophosphorylase has regulatory properties similarto those of the other plant ADPglucose pyrophosphorylases (Plaxton andPreiss 1987). They show that earlier reports claiming that the maizeendosperm ADPglucose pyrophosphorylase had enhanced activity in theabsence of activator (3-PGA) and decreased sensitivity to the inhibitor(P_(i)), was due to proteolytic cleavage of the enzyme during theisolation procedure. By altering an ADPglucose pyrophosphorylase gene toproduce an enzyme analogous to the proteolytically cleaved maizeendosperm ADPglucose pyrophosphorylase, decreased allosteric regulationwill be achieved.

To assay a liquid culture of E. coli for ADPglucose pyrophosphorylaseactivity, the cells are spun down in a centrifuge and resuspended inabout 2 ml of extraction buffer (0.05M glycylglycine pH 7.0, 5.0 mM DTE,1.0 mM EDTA) per gram of cell paste. The cells are lysed by passingtwice through a French Press. The cell extracts are spun in amicrocentrifuge for 5 minutes, and the supernatants are desalted bypassing through a G-50 spin column.

The enzyme assay for the synthesis of ADPglucose is a modification of apublished procedure (Haugen et al., 1976). Each 100 μl assay contains:10 μmole Hepes pH 7.7, 50 μg BSA, 0.05 μmole of [¹⁴C]glucose-1-phosphate, 0.15 μmole ATP, 0.5 μmole MgCl₂, 0.1 μg ofcrystalline yeast inorganic pyrophosphatase, 1 mM ammonium molybdate,enzyme, activators or inhibitors as desired, and water. The reactionmixture is incubated at 37° C. for 10 minutes, and is stopped by boilingfor 60 seconds. The assay is spun down in a microcentrifuge, and 40 μlof the supernatant is injected onto a Synchrom Synchropak AX-100 anionexchange HPLC column. The sample is eluted with 65 mM KPi pH 5.5.Unreacted [¹⁴ C]glucose-1-phosphate elutes around 7-8 minutes, and [¹⁴C]ADPglucose elutes at approximately 13 minutes. Enzyme activity isdetermined by the amount of radioactivity found in the ADPglucose peak.

The plant ADPGPP enzyme activity is tightly regulated, by both positive(3-phosphoglycerate; 3-PGA) and negative effectors (inorganic phosphate;P_(i)) (Ghosh and Preiss, 1966; Copeland and Preiss 1981; Sowokinos andPreiss 1982; Morell et al., 1987; Plaxton and Preiss, 1987; Preiss;1988;) and the ratio of 3-PGA:P_(i) plays a prominent role in regulatingstarch biosynthesis by modulating the ADPGPP activity (Santarius andHeber, 1965; Heldt et al., 1977; Kaiser and Bassham, 1979). The plantADPGPP enzymes are heterotetramers of two large/"shrunken" and twosmall/"Brittle" subunits (Morell et al., 1987; Lin et al., 1988a, 1988b;Krishnan et al., 1986; Okita et al., 1990) and there is strong evidenceto suggest that the heterotetramer is the most active form of ADPGPP.Support for this suggestion comes from the isolation of plant"starchless" routants that are deficient in either of the subunits (Tsaiand Nelson, 1966; Dickinson and Preiss, 1969; Lin et al., 1988a, 1988b)and from the characterization of an "ADPGPP" homotetramer of smallsubunits that was found to have only low enzyme activity (Lin et al.,1988b). In addition, proposed effector interaction residues have beenidentified for both subunits (Morell et al., 1988). Direct evidence forthe active form of the enzyme and further support of the kinetic datareported for the purified potato enzyme comes from the expression ofpotato ADPGPP activity in E. coli and the comparison of the kineticproperties of this material and that from potato tubers (Iglesias etal., 1993).

Unregulated enzyme variants of the plant ADPGPP are identified andcharacterized in a manner similar to that which resulted in theisolation of the E. coli glgC16 and related mutants such as glgC-SG5 andCL1136. A number of plant ADPGPP cDNA's, or portions of such cDNA's, forboth the large and small subunits, have been cloned from both monocotsand dicots (Anderson et al., 1989a; Olive et al., 1989; Muller et al.,1990; Bhave et al., 1990; du Jardin and Berhin, 1991; Smith-White andPreiss, 1992). The proteins encoded by the plant cDNA's, as well asthose described from bacteria, show a high degree of conservation (Bhaveet al., 1990). In particular, a highly conserved region, also containingsome of the residues implicated in enzyme function and effectorinteractions, has been identified (Morell et al., 1988; du Jardin andBerhin, 1991). Clones of the potato tuber ADPGPP subunit genes have beenisolated. These include a complete small subunit gene, assembled byaddition of sequences from the first exon of the genom/c clone with anearly full-length cDNA clone of the same gene, and an almost completegene for the large subunit. The nucleotide sequence (SEQ ID NO:7) andthe amino acid sequence (SEQ ID NO:8) of the assembled small subunitgene is given below. The nucleotide sequence presented here differs fromthe gene originally isolated in the following ways: a BglII+NcoI sitewas introduced at the ATG codon to facilitate the cloning of the geneinto E. coli and plant expression vectors by site directed mutagenesisutilizing the oligonucleotide primer sequence

GTTGATAACAAGATCTGTTAAC CATGGCGGCTTCC (SEQ ID NO: 11 ).

A SacI site was introduced at the stop codon utilizing theoligonucleotide primer sequence

CCAGTTAAAACGGAGCTCATCAGATGATGATTC (SEQ ID NO:12).

The SacI site serves as a 3' cloning site. An internal BglII site wasremoved utilizing the oligonucleotide primer sequence

GTGTGAGAACATAAATCTTGGATATGTTAC (SEQ ID NO:13).

This assembled gene was expressed in E. coli under the control of therecA promoter in a P recA-gene10L expression cassette (Wong et al.,1988) to produce measurable levels of the protein. An initiatingmethionine codon is placed by site-directed mutagenesis utilizing theoligonucleotide primer sequence

GAATTCACAGGGCCATGGCTCTAGACCC (SEQ ID NO: 14)

to express the mature gene.

The nucleotide sequence (SEQ ID NO:9) and the amino acid sequence (SEQID NO:10) of the almost complete large subunit gene is given below. Aninitiating methionine codon has been placed at the mature N-terminus bysite-directed mutagenesis utilizing the oligonucleotide primer sequence

AAGATCAAACCTGCCATGGCTTACTCTGTGATCACTACTG (SEQ ID NO:15). The purpose ofthe initiating methionine is to facilitate the expression of this largesubunit gene in E. coli. A HindIII site is located 103 bp after the stopcodon and serves as the 3' cloning site. The complete large ADPGPP geneis isolated by the 5' RACE procedure (Rapid Amplification of cDNA Ends;Frohman, 1990; Frohman et al., 1988; Loh et al., 1989). Theoligonucleotide primers for this procedure are as follows:

1) GGGAATTCAAGCTTGGATCCCGGGCCCCCCCCCCCCCCC (SEQ ID NO:16);

2) GGGAATTCAAGCTTGGATCCCGGG (SEQ ID NO:17); and

3) CCTCTAGACAGTCGATCAGGAGCAGATGTACG (SEQ ID NO:18).

The first two are the equivalent to the ANpolyC and the AN primers ofLoh et al. (1989), respectively, and the third is the reverse complementto a sequence in the large ADPGPP gone. The PCR 5' sequence products arecloned as EcoRI/HindIII/BamHI-PstI fragments and are easily assembledwith the existing gone portion.

The weakly regulated enzyme routants of ADPGPP are identified byinitially scoring colonies from a routagonized E. coli culture that showelevated glycogen synthesis, by iodine staining of 24-48 hour colonieson Luria-Agar plates containing glucose at 1%, and then bycharacterizing the responses of the ADPGPP enzymes from these isolatesto the positive and negative effectors of this activity (Cattaneo etal., 1969; Preiss et al., 1971). A similar approach is applied to theisolation of such variants of the plant ADPGPP enzymes. Given anexpression system for each of the subunit genes, muta-genesis of eachgone is carried out separately, by any of a variety of known means, bothchemical or physical (Miller, 1972) on cultures containing the gone oron purified DNA. Another approach is to use a PCR procedure (Ehrlich,1989) on the complete gone in the presence of inhibiting Mn++ions, acondition that leads to a high rate of misincorporation of nucleotides.A PCR procedure may also be used with primers adjacent to just aspecific region of the gone, and this mutagenized fragment then reclonedinto the non-mutagenized gene segments. A random syntheticoligonucleotide procedure may also be used to generate a highlymutagenized short region of the gone by mixing of nucleotides in thesynthesis reaction to result in misincorporation at all positions inthis region. This small region is flanked by restriction sites that areused to reinsert this region into the remainder of the gene. Theresultant cultures or transformants are screened by the standard iodinemethod for those exhibiting glycogen levels higher than controls.Preferably this screening is carried out in an E. coli strain deficientonly in ADPGPP activity and is phenotypically glycogen-minus and that iscomplemented to glycogen-plus by glgC. The E. coli strain should retainthose other activities required for glycogen production. Both genes areexpressed together in the same E. coli host by placing the genes oncompatible plasmids with different selectable marker genes, and theseplasmids also have similar copy numbers in the bacterial host tomaximize heterotetramer formation. An example of such an expressionsystem is the combination of pMON17335 and pMON17336 (Iglesias et al.,1993). The use of separate plasmids enables the screening of amutagenized population of one gone alone, or in conjunction with thesecond gone following transformation into a competent host expressingthe other gene, and the screening of two mutagenized populationsfollowing the combining of these in the same host. Followingre-isolation of the plasmid DNA from colonies with increased iodinestaining, the ADPGPP coding sequences are recloned into expressionvectors, the phenotype verified, and the ADPGPP activity and itsresponse to the effector molecules determined. Improved variants willdisplay increased V_(max), reduced inhibition by the negative effector(P_(i)), or reduced dependence upon activator (3-PGA) for maximalactivity. The assay for such improved characteristics involves thedetermination of ADPGPP activity in the presence of P_(i) at 0.045 mM(I₀.5 =0.045 mM) or in the presence of 3-PGA at 0.075 mM (A₀.5 =0.075mM). The useful variants will display <40% inhibition at thisconcentration of P_(i) or display >50% activity at this concentration of3-PGA. Following the isolation of improved variants and thedetermination of the subunit or subunits responsible, the mutation(s)are determined by nucleotide sequencing. The mutation is confirmed byrecreating this change by site-directed mutagenesis and reassay ofADPGPP activity in the presence of activator and inhibitor. Thismutation is then transferred to the equivalent complete ADPGPP cDNAgene, by recloning the region containing the change from the alteredbacterial expression form to the plant form containing the amyloplasttargeting sequence, or by site-directed mutagenesis of the completenative ADPGPP plant gene.

EXAMPLE 1 Construction of DNA Vectors for glgC16 Expression

To express the E. coli glgC16 gene in plant cells, and to target theenzyme to the plastids, the gene needed to be fused to a DNA encodingthe plastid-targeting transit peptide (hereinafter referred to as theCTP/ADP-glucose pyrophosphorylase gene), and to the proper plantregulatory regions. This was accomplished by cloning the glgC16 geneinto a series of plasmid vectors that contained the needed sequences.

The plasmid pLP226 contains the glgC16 gene on a HincII fragment, clonedinto a pUC8 vector at the HincII site (Leung et al. 1986). pLP226 wasobtained from Dr. Jack Preiss at Michigan State University, and wastransformed into frozen competent E. coli JM101 cells, prepared by thecalcium chloride method (Sambrook et al., 1989). The transformed cellswere plated on 2XYT (infra) plates that contained ampicillin at 100μg/ml. The plasmid pLP226 was purified by the rapid alkaline extractionprocedure (RAE) from a 5 ml overnight culture (Birnboim and Doly, 1979).

To fuse the glgC16 gene to the DNA encoding the chloroplast transitpeptide, a NcoI site was needed at the 5' end of the gene. A SacI sitedownstream of the termination codon was also needed to move theCTP/ADP-glucose pyrophosphorylase gene into the next vector. In order tointroduce these sites, a PCR reaction (#13) was run using approximately20 ng of rapid alkaline extraction-purified plasmid pLP226 for atemplate. The reaction was set up following the recommendations of themanufacturer (Perkin Elmer Cetus). The primers were QSP3 and QSP7. QSP3was designed to introduce the NcoI site that would include the startcodon for the glgC 16 gene. The QSP7 primer hybridized in the 3'nontranslated region of the glgC16 gene and added a SacI site. TheThermal Cycler was programmed for 30 cycles with a 1 min 94° C.denaturation step, a 2 min 50° C. annealing step, and a 3 min 72° C.extension step. After each cycle, the extension step was increased by 15sec. QSP3 Primer: 5' GGAGTTAGCCATGGTTAGTTTAGAG 3' (SEQ ID NO: 19) QSP7Primer:

5' GGCCGAGCTCGTCAACGCCGTCTGCGATTTGTGC 3' (SEQ ID NO: 20)

The PCR product was cloned into vector pGEM3zf+(Promega, Madison, Wis.),which had been digested with SacI and Hind III and had the DNA for themodified Arabidopsis small subunit CTP ligated at the HindIII site. TheDNA and amino acid sequences of this CTP are shown in SEQ ID NO: 5 andSEQ ID NO: 6, respectively.

The linearized vector was treated with 5 units of calf intestinalalkaline phosphatase for 30 min at 56° C. Then, both the vector and thePCR #13 fragment, which had the glgC16 gene with the new NcoI and SacIsites, were run on an agarose gel and the fragments were purified bybinding to DEAE membranes. The protocol used for the fragmentpurification with the DEAE membrane is from Schleicher and Schuell, andis titled "Binding and Recovery of DNA and RNA Using S and S DEAEMembrane."

Ligation #5 fused the glgC16 gene to the DNA for the modifiedArabidopsis SSU CTP with the pGEM3zf+. The ligation contained 3 μl ofvector that had been digested with NcoI and SacI, along with 3 μl of thePCR #13 product, that had also been cut with NcoI and SacI andrepurified on a gel. 5 μl (of 20 μl total) of ligation #5 wastransformed into frozen competent JM101 cells, and the transformed cellswere plated on 2XYT plates (16 g/l Bactotryptone, 10 g/l yeast extract,10 g/l NaCl, pH 7.3, and solidified with 1.5% agar) containingampicillin.

Sample 1 was picked from a plate after overnight growth. This sample wasinoculated into 4 ml of 2XYT media and grown overnight at 37 ° C. Theplasmid was isolated by the rapid alkaline extraction procedure, and theDNA was digested with EcoRI, NcoI, and EcoRI and NcoI together. Thedigest was separated on an agarose gel, and the expected fragments wereobserved. The plasmid isolated from sample 1 of was designatedpMON20100, and consisted of pGEM3zf+, the DNA for the modifiedArabidopsis SSU CTP, and the glgC16 gene. The fusion was in theorientation that allowed it to be transcribed from the SP6 polymerasepromoter.

To test this construct for import of the ADPglucose pyrophosphorylaseinto isolated lettuce chloroplasts, the CTP/ADPglucose pyrophosphorylasefusion needed to be transcribed and translated to produce [³⁵ S]-labeledADPglucose pyrophosphorylase. To make a DNA template for transcriptionby the SP6 polymerase, the CTP/ADPglucose pyrophosphorylase region ofpMON20100 was amplified by PCR to generate a large amount of linear DNA.To do this, about 0.1 μl of pMON20100, that had been purified by rapidalkaline extraction, was used as a template in PCR reaction #80. Theprimers were a commercially available SP6 promoter primer (Promega) andthe oligo QSP7 (SEQ ID NO:20). The SP6 primer hybridized to the SP6promoter in the vector, and included the entire SP6 promoter sequence.Therefore, a PCR product primed with this oligo will contain therecognition sequence for the SP6 polymerase. The QSP7 (SEQ ID NO:20)primer will hybridize in the 3' nontranslated region of the glgC16 gene.This is the same primer that was used to introduce a SacI sitedownstream of the glgC16 termination codon. The Thermal Cycler wasprogrammed for 30 cycles with a 1 min denaturation at 94° C., a 2 minannealing at 55° C., and a 3 min extension at 72° C. After each cycle,15 sec were added to the extension step. SP6 Promoter Primer: 5'GATTTAGGTGACACTATAG 3' (SEQ ID NO:21)

5 μl of PCR reaction #80 was run on an agarose gel and purified bybinding to DEAE membrane. The DNA was eluted and dissolved in 20 μl ofTE. 2 μl of the gel-purified PCR #80 product was used in an SP6 RNApolymerase in vitro transcription reaction. The reaction conditions werethose described by the supplier (Promega) for the synthesis of largeamounts of RNA (100 μl reaction). The RNA produced from the PCR reaction#80 DNA was used for in vitro translation with the rabbit reticulocytelysate system (Promega). ³⁵ S-labeled protein made from pMON20100 (i.e.,PCR reaction #80) was used for an in vitro chloroplast import assay aspreviously described. After processing the samples from the chloroplastimport assay, the samples were subjected to electrophoresis on SDS-PAGEgels with a 3-17% polyacrylamide gradient. The gel was fixed for 20-30min in a solution with 40% methanol and 10% acetic acid. Then, the gelwas soaked in EN3HANCE™ for 20-30 min, followed by drying the gel on agel dryer. The gel was imaged by autoradiography, using an intensifyingscreen and an overnight exposure. The results demonstrated that thefusion protein was imported into the isolated chloroplasts.

The construct in pMON20100 next was engineered to be fused to theenhanced CaMV 35S promoter (Kay, R. 1987) and the NOS 3' end (Bevan, M.1983) isolated from pMON999. PCR reaction 114 contained plasmidpMON20100 as a template, and used primers QSMll and QSM10. QSM11annealed to the DNA for the modified Arabidopsis SSU CTP and created aBglII site 7 bp upstream from the ATG start codon. QSM10 annealed to the3' end of the glgC16 gene and added an XbaI site immediately after thetermination codon, and added a SacI site 5 bp after the terminationcodon. The SacI site that had earlier been added to the glgC16 gene wasapproximately 100 bp downstream of the rex-ruination codon. The ThermalCycler was programmed for 25 cycles with a 1 min 94° C. denaturation, a2 min 55° C. annealing, and a 3 min 72° C. extension step. With eachcycle, 15 sec was added to the extension step.

QSM11 Primer (SEQ ID NO:22):

5' AGAGAGATCTAGAACAATGGCTTCCTCTATGCTCTCTTCCGC 3'

QSM10 Primer (SEQ ID NO:23):

5' GGCCGAGCTCTAGATTATCGCTCCTGTTTATGCCCTAAC 3'

95μl (from 100 μl total volume) of PCR reaction #114 was ethanolprecipitated, and resuspended in 20 μl of TE. 5 μl of this was digestedwith BglII (4 units) and SacI (10 units) overnight at 37° C. 5 μl (5 μg)of the vector, pMON999, which contains the enhanced CaMV 35S promoterand the NOS 3' end, was digested in the same manner. After digestionwith the restriction enzymes, the DNAs were run on an agarose gel andpurified by binding to DEAE membranes. Each of the DNAs were dissolvedin 20 gl of TE. 1 μl of PCR 114 was ligated with 3 μl of the vector, ina total volume of 20 μl. The ligation mixture was incubated at 14° C.for 7 hr. 10 μl of the ligation was transformed into frozen competentMM294 cells and plated on LB plates (10 g/l Bacto-tryptone, 5 g/l yeastextract, 10 g/l NaCl, and 1.5% agar to solidify) with 100/μg/mlampicillin. Colonies were picked and inoculated into tubes with 5 ml ofLB media with 100 μg/ml ampicillin, for overnight growth. The 5 mlovernight cultures were used for rapid alkaline extractions to isolatethe plasmid DNAs. The DNAs were digested with EcoRI, and separatealiquots were digested with NotI. After analyzing these samples onagarose gels, the plasmid pMON20102 was confirmed to have the 497 bpEcoRI fragment that is characteristic of the glgC16 gene. This plasmidalso contained the 2.5 kb NotI fragment which contained the enhancedCaMV 35S promoter, the DNA for the modified Arabidopsis SSU CTP, theglgC16 gene, and the NOS 3' end.

The pMON20102 plasmid was then used to construct a DNA vector whichwould express the glgC16 gene in a tuber-specific manner and would beused for the transformation of potato. This construct causes specificexpression of the ADPGPP in potato tubers and increases the level ofstarch in the tubers.

The vector used in the potato transformation is a derivative of theAgrobacterium mediated plant transformation vector pMON886. The pMON886plasmid is made up of the following well characterized segments of DNA.A 0.93 kb fragment isolated from transposon Tn7 which encodes bacterialspectinomycin/streptomycin (Spc/Str) resistance and is a determinant forselection in E. coli and Agrobacterium tumefaciens (Fling et al., 1985).This is joined to a chimeric kanamycin resistance gene engineered forplant expression to allow selection of the transformed tissue. Thechimeric gene consists of the 0.35 kb cauliflower mosaic virus 35Spromoter (P-35S) (Odell et al., 1985), the 0.83 kb neomycinphosphotransferase type II gene (NPTII), and the 0.26 kb3'-nontranslated region of the nopaline synthase gene (NOS 3') (Fraleyet al., 1983). The next segment is a 0.75 kb origin of replication fromthe RK2 plasmid (ori-V) (Stalker et al., 1981). It is joined to a 3.1 kbSalI to PvuI segment of pBR322 which provides the origin of replicationfor maintenance in E. coli (ori-322) and the bom site for theconjugational transfer into the Agrobacterium tumefaciens cells. Next isa 0.36 kb PvuI fragment from the pTiT37 plasmid which contains thehopaline-type T-DNA right border region (Fraley et al., 1985).

The glgC16 gene was engineered for expression primarily in the tuber byplacing the gene under the control of a tuber-specific promoter. TheGlgC16 protein was directed to the plastids within the plant cell due toits synthesis as a C-terminal fusion with a N-terminal protein portionencoding a chloroplast targeting sequence (CTP) derived from that fromthe SSU 1A gene from Arabidopsis thaliana (Timko et al., 1989). The CTPportion is removed during the import process to liberate the GlgC16enzyme. Other plant expression signals also include the 3'polyadenylation sequences which are provided by the NOS 3' sequenceslocated downstream from the coding portion of the expression cassette.This cassette was assembled as follows: The patatin promoter was excisedfrom the pBI241.3 plasmid as a HindIII-BamHI fragment (The pBI241.3plasmid contains the patatin-1 promoter segment comprising from the AccIsite at 1323 to the DraI site at 2289 [positions refer to the sequencein Bevan et al., 1986] with a HindIII linker added at the former and aBamHI linker added at the latter position; Bevan et al., 1986) andligated together with the CTP1-glgC16 fusion (the BgIII-SacI fragmentfrom pMON20102) and pUC-type plasmid vector cut with HindIII and SacI(these cloning sites in the vector are flanked by NotI recognitionsites). The cassette was then introduced, as a NotI site in pMON886,such that the expression of the glgC16 gene is in the same orientationas that of the NPTII (kanamycin) gene. This derivative is namedpMON20113, illustrated in FIG. 7 of Kishore, WO 91/19806.

Plant Transformation/Regeneration

The pMON20113 vector was mobilized into disarmed Agrobacteriumtumefaciens strain by the triparental conjugation system using thehelper plasmid pRK2013 (Ditta et al., 1980). The disarmed strain ABI wasused, carrying a Ti plasmid which was disarmed by removing thephytohormone genes responsible for crown gall disease. The ABI strain isthe A208 Agrobacterium tumefaciens carrying the disarmed pTiC58 plasmidpMP90RK (Koncz and Schell, 1986). The disarmed Ti plasmid provides thetrfA gene functions required for autonomous replication of the pMONvector after the conjugation into the ABI strain. When the plant tissueis incubated with the ABI::pMON conjugate, the vector is transferred tothe plant cells by the vir functions encoded by the disarmed pMP90RK Tiplasmid.

The pMON20113 construct, encoding the bacterial ADPGPP gene (SEQ IDNO:1), was transformed into the Russet Burbank potato variety Williamsby the following procedure. To transform Russet Burbank potatoes,sterile shoot cultures of Russet Burbank are maintained in sundae cupscontaining 8 ml of PM medium supplemented with 25 mg/L ascorbic acid(Murashige and Skoog (MS) inorganic salts, 30 g/l sucrose, 0.17 g/l NaH₂PO₄ H₂ O, 0.4 mg.l thiamine-HCl, and 100 mg/l myo-inositol, solidifiedwith 2 g/l Gelrite at pH 6.0). When shoots reach approximately 5 cm inlength, stem internode segments of 3-5 mm are excised and inoculatedwith a 1:10 dilution of an overnight culture of Agrobacteriumtumefaciens from a 4 day old plate culture. The stem explants areco-cultured for 2 days at 20° C. on a sterile filter paper placed over1.5 ml of a tobacco cell feeder layer overlaid on 1/10 P medium (1/10strength MS inorganic salts and organic addenda without casein as inJarret et al. (1980), 30 g/l sucrose and 8.0 g/l agar). Followingco-culture, the explants are transferred to full strength P-1 medium forcallus induction, composed of MS inorganic salts, organic additions asin Jarret et al. (1980), with the exception of casein, 5.0 mg/l zeatinriboside (ZR), and 0.10 mg/l naphthalene acetic acid NAA (Jarret et al.,1980a, 1980b). Carbenicillin (500 mg/l) and cefotaxime (100 mg/L) areincluded to inhibit bacterial growth, and 100 mg/l kanamycin is added toselect for transformed cells.

After 4 weeks, the explants are transferred to medium of the samecomposition, but with 0.3 mg/l gibberellic acid (GA3) replacing the NAA(Jarret et al., 1981) to promote shoot formation. Shoots begin todevelop approximately 2 weeks after transfer to shoot induction medium.These shoots are excised and transferred to vials of PM medium forrooting. After about 4 weeks on the rooting medium, the plants aretransferred to soil and are gradually hardened off. Shoots are testedfor kanamycin resistance conferred by the enzyme neomycinphosphotransferase II, by placing the shoots on PM medium for rooting,which contains 50 mg/L kanamycin, to select for transformed cells.

Russet Burbank Williams plants regenerated in culture were transplantedinto 6 inch (˜15.24 cm) pots and were grown to maturity under greenhouseconditions. Tubers were harvested and were allowed to suberize at roomtemperature for two days. All tubers greater than 2 cm. in length werecollected and stored at 3° C. under high humidity.

Specific Gravity and Starch Determinations of Stored Tubers

Specific gravity (SG) was determined after 3 and 4 months of cold (3 °C.) storage for the largest 2 or 3 tubers from each plant, with typicalweights being 20-40 grams per tuber. Tubers were allowed to warm to roomtemperature for a few hours prior to specific gravity determination, butwere not allowed to recondition. Specific gravity calculations wereperformed by the weight in air/weight in water method, where SG =weightin air/(weight in air-weight in water). Calculations for percent starchand percent dry matter based on SG were according to the followingformulas (yon Scheele, 1937):

    % starch=17.546+(199.07)(SG-1.0988)

    % dry matter=24.182+(211.04)(SG-1.0988)

Starch analysis was performed on fresh, center sections of stored tubertissue as described (Lin et al., 1988). Tubers were not allowed to warmbefore harvesting tissue. Briefly, approximately 100 mg. center sectionswere cut, weighed, placed in 1.5 ml centrifuge tubes, and frozen on dryice. The tissue was then dried to a stable weight in a Savant Speed-VacConcentrator, and final dry weight was determined. Soluble sugars werefirst removed by extracting three times with 1 ml. of 80% ethanol at 70° C., for 20 minutes per treatment. After the final incubation, allremaining ethanol was removed by desiccation in a Speed- VacConcentrator. The solid material was resuspended in 400 μl 10.2Mpotassium hydroxide, ground, and then incubated for 30 min. at 100 ° Cto solubilize the starch. The solutions were cooled and neutralized byaddition of 80 μl 1N acetic acid. Starch was degraded to glucose bytreatment with 14.8 units of pancreatic alpha-amylase (Sigma Chemical,St. Louis) for 30 min. at 37° C., followed by 10 units ofamyloglucosidase (Sigma Chemical, St. Louis) for 60 min. at 55 ° C.Glucose released by the enzymatic digestions was measured using theSigma (St. Louis) hexokinase kit, and these values were used tocalculate starch content.

Sugar Analysis

Tubers were stored at 3° C. and were not allowed to recondition at roomtemperature prior to sugar analysis. Center cuts from stored tubers wereobtained, fresh weights determined, and the tissue was frozen on dry iceprior to desiccation in Savant Speed-Vac Concentrator. Approximate freshweight per sample was 100 mg. Dry tuber material was coarsely ground,and sugars were extracted three times with 0.5 ml 80% ethanol at 70 ° C.for 20 minutes per extraction. After each incubation, the insolublematerial was spun down for 2 minutes in a microcentrifuge and thesupernatant collected. The supernatants from all three extractions werecombined, dried down, and resuspended in 1 ml 100 mM Tris buffer, pH7.5. For each sugar analysis, 10μl of sample was used.

For each sample, glucose content was determined using a Glucose [HK]diagnostic kit (Sigma Chemical Co., St. Louis, Mo.) according tomanufacturers protocol. Briefly, 1 mL of reconstituted reagent wasincubated with 10 μl of sample at room temperature for 10 minutes, andthe sample concentration determined by measuring absorbance at 340 nmsubtracting the absorbance of 10 μl of sample in water. Percent glucosewas then calculated by the equation:

    % glucose=[(A.sub.340 ×2.929)/mg. fresh weight]×100%

Fructose content was determined by adding 1μg of phosphoglucoisomeraseto the above reaction for glucose determination, and subtracting theresultant percent glucose+fructose value from percent glucose. Sucrosecontent was determined by addition of 1 μg phosphoglucoisomerase and 100μg yeast invertase to the glucose HK assay, and extending incubationtime to 30 minutes at room temperature. Percent sucrose was determinedas above, subtracting the values obtained for glucose and fructosecontent.

Western Blot Analysis of Stored Tubers

Tubers stored at 3 ° C. were not allowed to warm prior to isolation oftissue for analysis. For Western blot analysis, proteins were extractedfrom desiccated, coarsely powdered tuber tissue by grinding 1:1 in 100mM Tris pH 7.5, 35 mM KCl 5 mM dithiothreitol, 5 mM ascorbate, 1 mMEDTA, 1 mM benzamidine, and 20% glycerol. The protein concentration ofthe extract was determined using the Pierce BCA method, and proteinswere separated on 3-17% SDS polyacrylamide gels (Laemmli, 1970). E. coliADPGPP was detected using goat antibodies raised against purified E.coli ADPGPP and alkaline phosphatase conjugated rabbit anti-goatantibodies (Promega, Madison, Wis.).

Fry Color Determination

Eight transgenic potato lines expressing the E. coli glgC16 gene, and 20control lines consisting of a combination of lines from the pMON20113transformation event which do not express the E. coli glgC16 gene, andseveral nontransgenic Russet Burbank control lines, were grown underfield conditions in Parma, Id. Tubers were harvested and stored for twomonths at 40 ° C. Fry color was determined for all potato lines bytaking center cuts from representative samples from each line and fryingat 375 ° F. in soybean oil for 3 minutes and 30 seconds. Fry color wasdetermined by photovoltaic measurement and values were reportedaccording to the USDA color class chart for frozen french fries.

Results

All tubers were harvested from plants of the same variety (RussetBurbank Williams 82), the same age, and grown side by side underidentical growth conditions. Western blot analysis showed that levels ofE. coli ADPGPP were essentially equivalent to levels determined atharvest (Table 1), suggesting that the levels of E. coli ADPGPP proteinare stable during cold storage. Analysis of tubers stored at 3° C. underhigh humidity shows that those expressing the E. coli glgC16 geneaccumulate 5 - to 6-fold less reducing sugar than do control tubers(Tables 2, 3, 4, 5, and 6). Sucrose levels were comparable betweencontrol and transgenic tubers, while starch levels were significantlyhigher in the transgenic tubers. These results suggest that as starch isdegraded during storage, the sugars formed tend to be resynthesized intostarch in those tubers expressing the E. coli glgC16 gene, while incontrol tubers the sugars tend to accumulate.

Transgenic potato plants expressing the E. coli glgC16 gene have beengrown under field conditions and tubers from GlgC16 potato lines werestored at 40° F. (4° C.) along with tubers from several differentcontrol lines. Fry color, which directly correlated with sugar content,was determined after two months cold storage. The average fry color inthe transgenic potato tubers was significantly improved (lighter)relative to that in control tubers (darker color) stored under identicalconditions (Table 7), demonstrating that sugar levels were lower in thetubers expressing the E. coli glgC16 gene. Direct measurement ofreducing sugar content in a sample of the field grown tubers stored for14 weeks at 3° C. supports the fry color results in that tubersexpressing the E. coli glgC16 gene contained significantly less reducingsugar than controls (Table 8). Tubers from transgenic potato plants weretested for rate and degree of reconditioning following cold storage. Thefry color of transgenic lines which produce tubers having a specificgravity greater than 1.083 indicated an increased rate and degree ofreconditioning at 65° F. as compared to controls (Table 9).

                  TABLE 1                                                         ______________________________________                                        Expression of E. coli ADPGPP in potato tubers at harvest and after            3 months cold storage. E. coli ADPGPP levels were estimated from              Western blot analysis by comparison to known standards. Values are            given in ng GlgC16 per 50 μg extracted tuber protein.                                   ng GlgC16                                                        Line           Harvest 3 Months                                               ______________________________________                                        353c           20-25   20-25                                                  535c           25-30   20-25                                                  448a           25-30   25-30                                                  182a            2      0.5-1                                                  199a           20-25   20-25                                                  288c           20-25   20-25                                                  194a           15-20   15-20                                                  524a           10      15-20                                                  ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        Sugar and starch content (Dry Weight measurements) in 3 month                 cold stored tubers. Reducing sugars are glucose and fructose, and total       sugars are reducing sugars plus sucrose. Values (percent dry weight)          represent the averages from 9 glgC16 + high starch potato lines, and          11 control (glgC16-) potato lines stored for 3 months at 3° C.         Reducing Sugars  Sucrose Total Sugars                                                                            Starch                                     ______________________________________                                        glgC16+ 1.5          1.2     2.6     59.5                                     Control 7.0          0.8     7.8     53.7                                     ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        Sugar and starch content (Fresh Weight measurements) in 4 month               cold stored tubers. Reducing sugars are glucose and fructose, and total       sugars are reducing sugars plus sucrose. Values (percent fresh weight)        represent the average from glgC16 + high starch potato lines, and 11          control (glgC16-) potato lines stored for 4 months at 3° C.            Reducing Sugars  Sucrose Total Sugars                                                                            Starch                                     ______________________________________                                        glgC16+ 0.1          0.1     0.3     9.9                                      Control 0.8          0.2     1.0     6.0                                      ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        Reducing sugar content of potato tubers after 4 months cold storage.          Numbers of plant lines containing sugar levels within the ranges shown        are reported. Percentages are based on fresh weight.                                 Percent Reducing Sugar                                                        0-.2 .2-.4    .4-.6  .6-.8  .8-1.0                                                                              1.0+                                 ______________________________________                                        Control lines                                                                          0      2        0    4      2     3                                  glgC16 lines                                                                           6      3        0    0      0     0                                  ______________________________________                                    

                  TABLE 5                                                         ______________________________________                                        Total sugar content of potato tubers after 4 months cold storage.             Numbers of plant lines containing sugar levels within the ranges shown        are reported. Percentages are based on fresh weight.                                 Percent Total Sugars                                                          0-1  1-2      2-3    3-4    4-5  5+                                    ______________________________________                                        Control lines                                                                          0      0        0    2      3    6                                   glgC16 lines                                                                           3      4        2    0      0    0                                   ______________________________________                                    

                  TABLE 6                                                         ______________________________________                                        Starch content of potato tubers after 4 months cold storage. Numbers          of plant lines containing starch levels within the ranges shown are           reported. Percentages are based on fresh weight.                              Percent Starch                                                                2-4         4-6    6-8      8-10  10-12  12-14                                ______________________________________                                        Control lines                                                                         1       6      3      1     0      0                                  glgC16 lines                                                                          0       0      2      3     3      1                                  ______________________________________                                    

                  TABLE 7                                                         ______________________________________                                        Average fry color of field grown tubers after 2 months cold storage           at 40° F.. The fry color rating was assigned according to the          USDA                                                                          published color standards for frozen fried potatoes. In this rating, 0 =      very light color and 4 = very dark color. Numbers of plant lines having       fry colors within the ranges shown are reported.                                      Fry Color Rating                                                              2-2.49   2.5-2.99 3.0-3.49  3.5-4.0                                   ______________________________________                                        Control lines                                                                           0          0        4       16                                      glgC16 lines                                                                            1          1        6       0                                       ______________________________________                                    

                  TABLE 8                                                         ______________________________________                                        Reducing sugar content of field grown potato tubers after 14 weeks            storage at 3° C.. Numbers of plant lines containing sugar levels       within                                                                        the ranges shown are reported. Percentages are based on fresh weight.                    Percent Reducing Sugars                                                       0.5-1                                                                              1-1.5      1.5-2  2-2.5                                       ______________________________________                                        Control lines                                                                              0      4          2    2                                         glgC16 lines 4      2          2    0                                         ______________________________________                                    

EXAMPLE 2

Following storage at low temperatures, potatoes are frequentlyunacceptable for frying due to elevated sugar levels. These storedpotatoes are improved by treatments such as blanching or reconditioning;the former treatment removes sugars by treatment of the potato sliceswith hot water and in the latter the sugars are metabolized duringstorage of the tubers at higher temperatures (˜65 ° F.). Blanching isused to inactivate enzymes primarily but when sugars are high the timesemployed in this step are extended to many times the normal. Theextension of this step results in lower recovery of product, a loss offlavor, is time consuming, requires high energy input, and produceswaste material with high biological oxygen demand and thus posesadditional limitations on the disposal of the waste water.Reconditioning requires additional controlled temperature storagefacilities and optimal results may require a number of steps atdifferent temperatures. Sprouting and incidence of disease will increaseat the higher temperatures and with time. The fry color of fries madefrom cold-stored GlgC16 tubers were frequently lower (better) thancontrols and in some cases were low enough even after 3-4 months that noreconditioning would be required. These tests have been extended totubers stored for 2 months at 50° F. and then for 3 months at 38° F. andinclude a measure of the rate of reconditioning also.

Tubers from plants transformed with the following vectors were tested:pMON17316 (with the patatin 3.5 promoter) and pMON17279 (with the smallsubunit of potato ADPGPP); as well as tubers from plants containing thepatatin 1.0 promoter/glgC16 vector described above. These vectors wereconstructed as follows:

The patatin 3.5 promoter was obtained from the plasmid pPBI240.7 (Bevan,1986). The majority of the 3.5 promoter was excised from pPBI240.7, fromthe HindIII site (-3500) to the XbaI site at -337, and combined with theremainder of the promoter, from the XbaI site to a BglII site at +22(formerly a DraI site), in a triple ligation into a vector whichprovided a BglII site to form pMON17280. This latter plasmid then servedas the vector for the triple ligation of the complete 3.5 promoter andthe plastid target peptide-GlgC16 fusion from pMON20102, described aboveto form the tuber expression cassette (in pMON17282). This cassette,consisting of the patatin 3.5 promoter, the plastid targetpeptide-GlgC16 fusion, and the NOS 3' sequences, was introduced into theplant transformation vector pMON17227, a Ti plasmid vector disclosed anddescribed by Barry et al. in WO 92/04449 (1991), incorporated herein byreference, on a NotI fragment to form pMON17316. See FIG. 1.

The promoter for the potato tuber ADPGPP small subunit gene, SEQ IDNO:24, was obtained as a XbaI-BglII fragment of the genomic clone 1-2and inserted into the XbaI and BamHI site of Bluescript II KS- (Nakataet al., 1992). The promoter fragment used consists of the portion fromthe ClaI site about 2.0 kb 5' from the putative initiation methionineand extending to the HindIII site located 12 bp before this ATG. A BglIIsite was placed adjacent to this HindIII site by subcloning throughanother pUC vector, and was linked through this latter site to thefusion of the CTP targeting and the glgC16 coding sequences. Thiscassette, with a plant 3' recognition sequence was cloned into planttransformation vectors to form pMON17279 (also includes a cassette inwhich the E. coli uidA [GUS] gene is expressed from the same smallpotato 5 ADPGPP promoter). See FIG. 2.

These vectors were inserted into potato cells by Agrobacteriumtransformation followed by glyphosate selection. To transform potatoesusing glyphosate as a selectable agent, the appropriate Agrobacteriumwas grown overnight in 2 ml of LBSCK. The following day, the bacteriawas diluted 1:10 with MSO or until an optical density reading of0.2-0.33 was established. Leaves from the stems of potato plants thathad been grown under sterile conditions for three weeks on PM mediasupplemented with 25 mg/ml ascorbic acid were removed, stems were cutinto 3-5 mm segments and inoculated with diluted bacteria as describedpreviously. Explants were placed onto prepared co-culture plates. Theco-culture plates contained 1/10 MSO with 1.5 mL of TxD cells overlainwith wetted filter paper. About 50 explants were placed per plate. After2 days co-culture period, explants were placed onto callus inductionmedia which contains 5.0 mg/l Zeatin Riboside, 10 mg/l AgNO3 and 0.1mg/l NAA for 2 days. Explants were subsequently transferred onto callusinduction media which contained 0.025 mM glyphosate for selection. After4 weeks, explants were placed onto shoot induction media which contained5.0 mg/l Zeatin Riboside+10 mg/l AgNO3 and 0.3 mg/l GA3, with 0.025 mMglyphosate for selection. Shoots began to appear at 8 weeks. Explantswere transferred to fresh shoot induction media every 4 weeks for 12weeks. Shoots were excised and placed on PM media for about 2 weeks oruntil they were large enough to be placed into soil.

The data for GlgC16 Russet Burbank lines, including those expressingGlgC16 from the patatin 1.0 (HS01; HS03; MT01), patatin 3.5 (HS13), andthe small subunit of potato ADPGPP (HS10) promoters is presented below(Table 9). A number of GlgC16 potato (variety Atlantic) lines were alsoexamined (MT01; patatin 1.0 promoter). A processor would typically haveto blanch to make acceptable products at a score of 2.0 or above. Anumber of Russet Burbank GlgC16 lines gave a fry score less than 2.0immediately out of cold storage and thus could be processed directly. Afry color score of less than 2.0 is obtained with a large number of thelines after a very short period of reconditioning. This improvedreconditioning response is seen for lines with increased solids and alsofor GlgC16 lines that did not show an increase in specific gravity. Theimprovement is also shown with all of these promoters used to expressGlgC16 in the tuber. The effect of obtaining lines that may be frieddirectly out of storage and that recondition rapidly is also shown forGlgC16 Atlantic. Line MT01-27 also demonstrated that increased starch inthe tuber is not necessary to obtain enhanced cold storage propertiessince the specific gravity of the lines tested was not significantlydifferent from that of the Atlantic control.

                  TABLE 9                                                         ______________________________________                                        Fry Color/Reconditioning response of potatoes containing GlgC16. (Fry         color rated according to USDA chart on a scale of 0-4; lowest-highest).                    Number of days at 65° F.                                  Line           0     3       6   10    13  17                                 ______________________________________                                        Control - Russet Burbank                                                      RB02           2.2   2.3     1.8 2.0   1.0 1.3                                RB03           3.5   3.3     2.5 1.7   2.3 2.5                                RB05           2.3   2.5     2.2 2.0   1.2 1.3                                GlgC16 Russet Burbank                                                         HS13-47*       3.0   2.5     0.7 0.7   1.3 1.3                                HS10-15*       1.3   1.3     0.4 0.8   1.0 1.0                                HS13-50*       3.2   1.0     0.5 1.0   1.2 0.8                                MT01-82*       2.2   1.2     0.7 1.0   1.5 1.0                                HS13-36*       2.2   1.3     0.7 1.2   1.2 0.8                                HS10-20*       1.0   0.7     0.5 0.5   0.4 0.5                                *-specific gravity greater than 1.083                                         HS01-58#       2.8   2.5     1.3 0.7   1.5 1.8                                HS03-20#       3.2   2.3     1.7 2.8   1.8 2.0                                HS03-18#       2.0   1.8     1.7 0.7   1.3 1.3                                HS03-12A#      3.3   2.5     2.7 2.2   2.2 0.8                                HS03-12B#      3.0   3.2     2.0 2.7   1.3 2.0                                HS01-49#       2.2   1.3     2.3 2.2   1.5 0.8                                HS13-30#       3.2   2.3     2.0 2.8   1.7 3.0                                #-specific gravity less than 1.083                                            Control - Atlantic                                                            AT-1           2.3   2.3     2.3 1.5   1.5 1.8                                GlgC16 Atlantic                                                               MT01-24        2.5   1.8     2.8 1.2   2.2 1.3                                MT01-27        1.0   0.5     1.3 0.3   0.8 0.7                                ______________________________________                                    

EXAMPLE 3

The effect of GlgC16 on delaying sprouting was tested on a population oftubers stored at 60° F. (these tubers had been stored previously at38°-40° F. for 3 months). The tubers (4-6) were examined at intervalsand scored for the presence of sprouts of >0.5 cm (Table 10). The delayin sprouting, represented as the number of days to 50% sprouted, wasfrequently improved in the GlgC16 lines, was observed in the threevarieties tested (Russet Burbank, Atlantic, and Norchip); was observedin lines where GlgC16 was expressed from the patatin 1.0 (HS01, HS03,and MT01), the patatin 3.5 (HS13), and the potato small ADPGPP (HS10)promoters; and was seen in lines with and without increased solidscontent. The lines with the delay sprouted normally when planted insoil.

                  TABLE 10                                                        ______________________________________                                        Variety                                                                              Line     Number of days to 50% sprouted                                ______________________________________                                        Russet Control  15                                                            Burbank                                                                              Control  14                                                                   Control  9                                                                    HS01-25  11                                                                   HS01-49  15                                                                   HS01-58  18                                                                   HS03-3   12                                                                   HS03-5   13                                                                   HS03-17  13                                                                   HS03-26  14                                                                   HS03-27  12                                                                   HS03-41  24                                                                   HS10-10  14                                                                   HS10-15  26                                                                   HS10-20  >43†                                                          HS13-2   11                                                                   HS13-13  16                                                                   HS13-23  23                                                                   HS13-30  15                                                                   HS13-34  25                                                                   HS13-37  12                                                                   HS13-47  21                                                                   HS13-50  19                                                                   HS13-68  13                                                                   HS13-70  24                                                                   MT01-10  14                                                                   MT01-11  13                                                                   MT01-30  14                                                                   MT01-37  19                                                                   MT01-82  16                                                            Atlantic                                                                             Control  6                                                                    MT01-6   11                                                                   MT01-7   9                                                                    MT01-15  10                                                                   MT01-31  6                                                             Norchip                                                                              Control  8                                                                    MT01-1   12                                                                   MT01-5   13                                                            ______________________________________                                         †duration of observation; tubers from this line sprouted when          planted in soil.                                                         

EXAMPLE 4

Additional tests were performed with potatoes transformed with glgC16under the control of two different promoters. Promoters for the largesubunit of potato tuber ADPGPP were isolated from two varieties ofpotato, Russet Burbank (SEQ ID NO:25) and Desiree (SEQ ID NO:26). Theclones were identified using plaque hybridization with a probe from the5' end of a cDNA from the large subunit of ADPglucose pyrophosphorylase.The translational start sites (ATG) of these clones were identified byplant consensus (Lutcke et al., 1987). PCR primers were used tointroduce an BAMHI site at the 3' end downstream of the ATG and aHINDIII site at the 5' end of both promoters. The resulting 600 bpRusset Burbank promoter and 1600 bp Desiree promoters were ligatedindependently into pMON10098 in place of the E35S promoter, and fusedwith a BglII-SacI fragment from pMON20102 containing CTP-glgC16 chimericgene. The E35S-NPTII-Nos cassette was removed from these plasmids andreplaced with a NotI- SalI fragment containing the FMV-CTP-CP4-E9cassette of pMON17227, discussed above, resulting in pMON21522 (RussetBurbank-derived promoter) and pMON21523 (Desiree-derived promoter). ThepMON10098 plasmid contains the following DNA regions: 1) The chimerickanamycin resistance gene engineered for plant expression to allowselection of the transformed tissue. The chimeric gene consists of the0.35 Kb cauliflower mosaic virus 35S promoter (P-35S) (Odell et al.,1985), the 0.83 Kb NPTII gene, and the 0.26 Kb 3'-nontranslated regionof the NOS 3'; 2) The 0.45 Kb ClaI to the DraI fragment from thepTi15955 octopine Ti plasmid, which contains the T-DNA left borderregion (Barker et al., 1983); 3) The 0.75 Kb segment containing theorigin of replication from the RK2 plasmid (ori-V) (Stalker et al.,1981); 4) The 3.0 Kb SalI to PstI segment of pBR322 which provides theorigin of replication for maintenance in E. coli (ori-322), and the bomsite for the conjugational transfer into the Agrobacterium tumefacienscells; 5) The 0.93 Kb fragment isolated from transposon Tn7 whichencodes bacterial spectinomycin/streptomycin resistance (Spc/Str) (Flinget al., 1985), and is a determinant for selection in E. coli andAgrobacterium tumefaciens; 6) The 0.36 Kb PvuI to BclI fragment from thepTiT37 plasmid, which contains the nopaline-type T-DNA right borderregion (Fraley et al., 1985); and 7) The last segment is the expressioncassette consisting of the 0.65 Kb cauliflower mosaic virus (CaMV) 35Spromoter enhanced by duplication of the promoter sequence (P-E35S) (Kayet al., 1987), a synthetic multilinker with several unique cloningsites, and the 0.7 Kb 3' nontranslated region of the pea rbcS-E9 gene(E9 3') (Coruzzi et al., 1984). The plasmid was mated into Agrobacteriumtumefaciens strain ABI, using the triparental mating system, and used totransform Russet Burbank line Williams 82.

The improvements in storage characteristics have also been shown forRusset Burbank transformed with pMON22152 and pMON21523, in which GlgC16is expressed from promoters for the large subunit of potato tuberADPGPP. Field grown tubers were stored initially,after harvest for 1month at 50° F., after which they were placed in cold storage at 40° F.for 4 months. In one test, the fry color of fries produced from thesetubers directly out of storage was evaluated by determining thereflectance of the fried material; lower values are preferred. In secondtest, a portion of the cold stored tubers were transferred to 55° F. todetermine the response in reconditioning. The data for theseevaluations, for both the stem and bud ends of the tubers are presentedin Table 11. In both cases, many lines have better color values than thecontrols, both for direct frying and following reconditioning. Forinstance, pMON21522-144, pMON21523-79, and many others show dramaticimprovements over the controls.

                  TABLE 11                                                        ______________________________________                                                Reflectance of fried strips.sup.1,2                                           40° F. Storage.sup.3                                                             40° F. Storage: 21 d.@ 55° F..sup.4           LINE #    Bud.sup.5                                                                             Stem.sup.5                                                                            Bud      Stem                                       ______________________________________                                        pMON21522                                                                     144       24.3    22.1    34.6     25.4                                       209       22.8    20.1    31.4     23.2                                       149       27.1    22.4    30.7     27.5                                       178       25.3    21.1    34.9     29.3                                       194       23.7    20.8    30.6     23.5                                       204       21.3    14.7    33.9     23.0                                       218       22.8    18.9    33.3     20.7                                       Control/Mean.sup.6                                                                      19.7    16.8    31.0     25.1                                       pMON21523                                                                     33        22.1    16.5    29.8     23.1                                       34        21.2    18.2    32.1     27.3                                       38        24.0    23.2    28.3     23.6                                       40        24.4    15.3    31.5     26.2                                       47        22.0    15.6    34.9     27.7                                       48        23.0    19.3    31.1     24.9                                       79        24.4    19.7    35.8     29.7                                       80        21.8    20.5    32.7     25.5                                       93        20.7    17.4    31.1     23.2                                       99        19.8    17.1    30.6     23.4                                       31        22.1    21.6    30.2     24.7                                       35        20.0    16.8    34.5     28.4                                       37        20.6    18.5    34.2     27.2                                       39        18.0    15.8    33.1     24.4                                       42        19.2    15.1    31.9     24.1                                       43        23.9    20.7    32.1     22.0                                       60        20.7    16.3    32.4     20.3                                       64        22.3    16.1    30.0     23.0                                       71        21.8    20.9    33.1     24.7                                       76        22.8    21.5    28.5     25.0                                       81        16.5    16.0    29.1     22.7                                       92        15.3    15.0    30.3     23.5                                       Control/Mean.sup.6                                                                      19.7    16.8    31.0     25.1                                       ______________________________________                                         .sup.1 Four central strips were cut from each of 4-6 tubers and fried at      375° F.                                                                .sup.2 Reflectance measurements were taken using a PHOTOVOLT 577              Reflectance meter.                                                            .sup.3 Strips were prepared from fieldgrown tubers that had been stored a     50 ° F. for 1 month and then at 40° F. for 4 months (cold       storage)                                                                      .sup.4 Strips were prepared from fieldgrown tubers that had been stored a     50 ° F. for 1 month and at 40° F. for 4 months (cold            storage), and subsequently reconditioned at 55° F. for 21 days.        .sup.5 Reflectance was measured separately on bud and stem ends of fried      strips.                                                                       .sup.6 The mean values for 22 control Russet Burbank lines are presented      for comparison.                                                          

From the foregoing, it will be seen that this invention is one welladapted to attain all the ends and objects hereinabove set forthtogether with advantages which are obvious and which are inherent to theinvention. It will be understood that certain features andsubcombinations are of utility and may be employed without reference toother features and subcombinations. This is contemplated by and iswithin the scope of the claims. Since many possible embodiments may bemade of the invention without departing from the scope thereof, it is tobe understood that all matter herein set forth or shown in theaccompanying drawings is to be interpreted as illustrative and not in alimiting sense.

REFERENCES

Anderson, et al. (1989a) J. Biol. Chem. 264 (21):12238-12242.

Anderson, et al. (1989b) First International Symposium on the MolecularBiology of the Potato, Bar Harbor, Me. ap Rees, et al. (1988) Syrup.Soc. Exp. Biol. 42: 377-393.

Baker, S.S., et al. (1994) Plant Mol. Biol. 24:701-713.

Barker, et al. (1983) Plant Mol. Biol. 2:335-350.

Bevan, et al. (1983) Nature (London) 304:184-187.

Bevan, et al. (1986) Nucleic Acids Res. 14 (11):4625-4638.

Bhave, et al. (1990) Plant Cell 2: 581-588.

Birnboim, et al. (1979) Nucleic Acids Res. 7:1513-1523.

Blennow, et al. (1991) Phytochemistry 30:437-444.

Burton, W. G. (1989) The Potato. Longman Scientific and Technical,Harlow, England, pp 865-522

Cattaneo, et al. (1969) Biochem. Biophys. Res. Commun. 34 (5):694-701.

Chang, et al. (1978) J. Bacteriol. 134; 1141-1156.

Claassen, et al. (1991)Plant Physiol. 95:1243-1249

Coffin, et al. (1987) J. Food Sci. 52: 639-645.

Copeland, L. and J. Preiss (1981) Plant Physiol. 68: 996-1001.

Coruzzi, et al. (1984) EMBO J. 3(8): 1671-1679.

Creuzet-Sigal, et al. (1972) "Genetic Analysis and BiochemicalCharacterization of Mutants Impairing Glycogen Metabolism in Escherichiacoli K12" in Biochemistry of the Glycosidic Linkage: An Integrated View.Edited by R. Piras and H. G. Pontis. 647-680. New York: Academic PressInc.

Dickinson, D. B. and J. Preiss (1969) Plant Physiol. 44:1058-1062.

Ditta, et al. (1980) Proc Natl Acad Sci USA 77, 7347-7351.

Dixon, et al. (1981) Phytochemistry 20: 969-972.

du Jardin, P. and Berhin, A. (1991) Plant Mol. Biol. 16:349-351.

Ebbelaar, et al. (1993) Int. Syrup. on Gen. Manip. of Plant Metabolismand Growth, 29-31 March, Norwich UKAbstract #9.

Ehrlich, H. A. (1989)Ed. PCR Technology--Principles and Apnlications forDNA Amplification. Stockton Press, New York.

Fling, et al. (1985) Nucleic Acids Research 13 no. 19, 7095-7106.

Fraley, et al. (1983) Proc Natl Acad Sci USA 80, 4803-4807.

Fraley, et al. (1985) Bio/Technology 3, 629-635.

Frohman, M. A. (1990) In: PCR Protocols. Innis, M. A., Gelfand, D. H.,Snincky, J. J., and White, T. J., eds. Academic Press, San Diego, Calif.pp. 28-38.

Frohman, et al. (1988) Proc. Natl. Acad. Sci. USA 85: 8998-9002.

Ghosh, et al. (1966) J. Biol. Chem. 241 (19):4491-4504.

Gilmour, et al. (1992) Plant Mol. Biol. 18:13-21.

Hajela, et al. (1990) Plant Physiol. 93:1246-1252.

Hannapel, D. J. (1990) Plant Physiol. 94: 919-925.

Hardenburg, et al. (1986) USDA Agric. Handbook No 66, pp 66-68

Haugen, et al (1976) J. Biol. Chem. 251. (24) 7880-7885

Heldt, et al. (1977) Plant Physiol. 59: 1146-1155.

Houde, et al. (1992) Plant Physiol. 99(4):1381-1387.

Huang, R.C., et al. (1987) FED Proc. 46 (6): 2238.

Iglesias, et al., (1993) J. Biol Chem. 268:1081-1086.

Jarret, et al. (1980a) Physiol. Plant. 49: 177-184.

Jarret, et al. (1980b) J. Amer. Soc. Hort. Sci. 105: 238-242.

Jarret, et al. (1981) In Vitro 17: 825-830.

Jefferson, et al. 1990. Plant Mol. Biol. 14: 995-1006.

Kaiser, W. M. and J. A. Bassham (1979) Plant Physiol. 63: 109-113.

Kay, et al. (1987) Science 236:1299-1302.

Koncz, C. and Schell, J. (1986) Mol. Gen. Genet. 204:383-396.

Kossmann et al. (1991) Mol. Gem Genet. 230:39-44.

Krishnan, et al (1986) Plant Physiol. 81: 642-645.

Kruger, N. J. and Hammond, J. B. W. (1988) Plant Physiol. 86: 645-648.

Kurkela, et al. (1990) Plant Mol. Biol. 15:137-144.

Laemmli, U. K. (1970) Nature (London) 227:680-685.

Leung, et al. (1986) J. Bact. 167 (1):82-88.

Lin, et al. (1988a) Plant Physiol. 86:1131-1135.

Lin, et al. (1988b) Plant Physiol. 88:1175-1181.

Liu, X. J., et al. (1990) Mol. Gen. Genet. 223:401-406.

Lob, et al. (1989) Science 243:217-220.

Lutcke et al. (1987) EMBO J. 6(1):43-48.

Mignery, et al. (1988) Gene Gene 62:27-44.

Miller, J. H. (1972) Experiments in Molecular Genetics. Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y.

Miner, et al. (1992) J. Neurosci. Res. 33(1): 10-18.

Morell, et al. (1988) J. Biol. Chem. 263:633-637.

Morell, et al. (1987) Plant Physiol. 85:182-187.

Mori et al. (1991) J. Biol. Chem. 266: 18446-18453.

Muller, et al. (1990) Mol. Gen. Genet. 224:136-146.

Murata, et al. (1992) Nature 356:710-713.

Nakano et al. (1989) J. Blochem. 106: 691-695.

Nakata, et al. (1992) J. Cell. Biochem. Suppl. 16F, Abst. Y311, p.266.

Nordin, K, et al. (1993) Plant Mol. Biol. 21: 641-653.

Odell, et al. (1985) Nature 313,810-812.

Ohta et al. (1991) Mol. Gen. Genet. 225: 369-378.

Okita, et al. (1990) 93: 785-790.

Olive, et al. (1989) Plant Mol. Biol. 12: 525-538.

Plaxton, et al. (1987) Plant Physiol. 83: 105-112.

Preiss, Jack. (1988) "Biosynthesis of Starch and Its Regulation" in TheBiochemistry of Plants. Edited by J. Preiss. 184-249. Orlando, Fla.:Academic Press.

Preiss, et al. (1971) Biochem. Biophys. Res. Comm. 42:180-186.

Pressey, R. (1966)Arch. Biochem. Biophys. 113: 667-674.

Qoronfieh, et al. (1992)J. Bacteriol. 174(24): 7902-7909.

Rocha-Sosa, et al. (1989) EMBO J. 8(1):23-29.

Rohde et al., (1990) J. Genet. & Breed. 44:311-315.

Ryall, A. L. and Lipton, W. J. (1979) Vegetables and Melons, Vol. 1.AVI, Westport, Conn., pp 225-230, 272-279.

Sambrook, et al. (1989) Molecular Cloning, A Laboratory Manual. 2nd Ed.Cold Spring Harbor Laboratory Press, N.Y.

Santarius, et al. (1965) Biochim. Biophys. Acta 102:39-54.

Shahar et al. (1992) Plant Cell 4:135-147.

Shallenberger, et al. (1959) Agric. and Food Chem. 7: 274-277.

Smith-White, B. and J. Preiss (1992) J. Mol. Evol. 34:449-464.

Solanoubat, M. and G. Belllard (1987) Geneg 60:47-56.

Solanoubat, M. and G. Belllard (1989) Geneg 84:181-185.

Sowokinos, J. R., and J. Preiss. (1982) Plant Physiol. 69:1459-1466.

Stalker, et al (1981) Mol Gert Genet 181, 8-12.

Stiekema et al. (1988) Plant Mol. Biol. 11: 255-269.

Stukerlj et al. (1990) Nucl. Acids Res. 18:46050.

Svab, et al. (1990) Proc. Natl. Acad. Sci. USA 87:8526-8530.

Takaha et al., (1993) J. Biol. Chem. 26 8:1391-1396.

Timko, et al. (1985) Nature (London) 318: 579-582.

Tsai, C. Y. and O. E. Nelson. (1966) Science 151: 341-343.

Vasil, V., F. Redway and I. Vasil. (1990) Bio/Technology 8:429-434.

van Berkel, J., et al. (1991) Abstract #1576 presented at theInternational Congress of Plant Molecular Biology, Tucson, Ariz.

van Berkel, J., et al. (1994) Plant Physiol. 104: 445-452.

Von Scheele, C. (1937) Landw Ver Stn 127: 67-96.

Weaver, et al. (1978) Am. Pot. J. 55:83-93.

White, T. C., et al. (1992) Plant Physiol. 99 (Suppl. 1):78.

Wilhelm, K. S., et al. (1993) Plant Mol. Biol. 23: 1073-1077.

Wong, et al. (1988) Geneg 68: 193-203.

Yamaguchi-Shinozaki, et al. (1993) Mol. Gem Genet. 238:331-340.

Yamaguchi-Shinozaki, K. and Shinozaki, K. (1994) The Plant Cell 6:251-264.

Yoshida et al. (1992) Geneg 10: 255-259.

Zhu, B., et al. (1993) Plant Mol. Biol. 21: 729-735.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 26                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1296 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..1296                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       ATGGTTAGTTTAGAGAAGAACGATCACTTAATGTTGGCGCGCCAGCTG48                            MetValSerLeuGluLysAsnAspHisLeuMetLeuAlaArgGlnLeu                              151015                                                                        CCATTGAAATCTGTTGCCCTGATACTGGCGGGAGGACGTGGTACCCGC96                            ProLeuLysSerValAlaLeuIleLeuAlaGlyGlyArgGlyThrArg                              202530                                                                        CTGAAGGATTTAACCAATAAGCGAGCAAAACCGGCCGTACACTTCGGC144                           LeuLysAspLeuThrAsnLysArgAlaLysProAlaValHisPheGly                              354045                                                                        GGTAAGTTCCGCATTATCGACTTTGCGCTGTCTAACTGCATCAACTCC192                           GlyLysPheArgIleIleAspPheAlaLeuSerAsnCysIleAsnSer                              505560                                                                        GGGATCCGTCGTATGGGCGTGATCACCCAGTACCAGTCCCACACTCTG240                           GlyIleArgArgMetGlyValIleThrGlnTyrGlnSerHisThrLeu                              65707580                                                                      GTGCAGCACATTCAGCGCGGCTGGTCATTCTTCAATGAAGAAATGAAC288                           ValGlnHisIleGlnArgGlyTrpSerPhePheAsnGluGluMetAsn                              859095                                                                        GAGTTTGTCGATCTGCTGCCAGCACAGCAGAGAATGAAAGGGGAAAAC336                           GluPheValAspLeuLeuProAlaGlnGlnArgMetLysGlyGluAsn                              100105110                                                                     TGGTATCGCGGCACCGCAGATGCGGTCACCCAAAACCTCGACATTATC384                           TrpTyrArgGlyThrAlaAspAlaValThrGlnAsnLeuAspIleIle                              115120125                                                                     CGTCGTTATAAAGCGGAATACGTGGTGATCCTGGCGGGCGACCATATC432                           ArgArgTyrLysAlaGluTyrValValIleLeuAlaGlyAspHisIle                              130135140                                                                     TACAAGCAAGACTACTCGCGTATGCTTATCGATCACGTCGAAAAAGGT480                           TyrLysGlnAspTyrSerArgMetLeuIleAspHisValGluLysGly                              145150155160                                                                  GTACGTTGTACCGTTGTTTGTATGCCAGTACCGATTGAAGAAGCCTCC528                           ValArgCysThrValValCysMetProValProIleGluGluAlaSer                              165170175                                                                     GCATTTGGCGTTATGGCGGTTGATGAGAACGATAAAACTATCGAATTC576                           AlaPheGlyValMetAlaValAspGluAsnAspLysThrIleGluPhe                              180185190                                                                     GTGGAAAAACCTGCTAACCCGCCGTCAATGCCGAACGATCCGAGCAAA624                           ValGluLysProAlaAsnProProSerMetProAsnAspProSerLys                              195200205                                                                     TCTCTGGCGAGTATGGGTATCTACGTCTTTGACGCCGACTATCTGTAT672                           SerLeuAlaSerMetGlyIleTyrValPheAspAlaAspTyrLeuTyr                              210215220                                                                     GAACTGCTGGAAGAAGACGATCGCGATGAGAACTCCAGCCACGACTTT720                           GluLeuLeuGluGluAspAspArgAspGluAsnSerSerHisAspPhe                              225230235240                                                                  GGCAAAGATTTGATTCCCAAGATCACCGAAGCCGGTCTGGCCTATGCG768                           GlyLysAspLeuIleProLysIleThrGluAlaGlyLeuAlaTyrAla                              245250255                                                                     CACCCGTTCCCGCTCTCTTGCGTACAATCCGACCCGGATGCCGAGCCG816                           HisProPheProLeuSerCysValGlnSerAspProAspAlaGluPro                              260265270                                                                     TACTGGCGCGATGTGGGTACGCTGGAAGCTTACTGGAAAGCGAACCTC864                           TyrTrpArgAspValGlyThrLeuGluAlaTyrTrpLysAlaAsnLeu                              275280285                                                                     GATCTGGCCTCTGTGGTGCCGAAACTGGATATGTACGATCGCAATTGG912                           AspLeuAlaSerValValProLysLeuAspMetTyrAspArgAsnTrp                              290295300                                                                     CCAATTCGCACCTACAATGAATCATTACCGCCAGCGAAATTCGTGCAG960                           ProIleArgThrTyrAsnGluSerLeuProProAlaLysPheValGln                              305310315320                                                                  GATCGCTCCGGTAGCCACGGGATGACCCTTAACTCACTGGTTTCCGGC1008                          AspArgSerGlySerHisGlyMetThrLeuAsnSerLeuValSerGly                              325330335                                                                     GGTTGTGTGATCTCCGGTTCGGTGGTGGTGCAGTCCGTTCTGTTCTCG1056                          GlyCysValIleSerGlySerValValValGlnSerValLeuPheSer                              340345350                                                                     CGCGTTCGCGTGAATTCATTCTGCAACATTGATTCCGCCGTATTGTTA1104                          ArgValArgValAsnSerPheCysAsnIleAspSerAlaValLeuLeu                              355360365                                                                     CCGGAAGTATGGGTAGGTCGCTCGTGCCGTCTGCGCCGCTGCGTCATC1152                          ProGluValTrpValGlyArgSerCysArgLeuArgArgCysValIle                              370375380                                                                     GATCGTGCTTGTGTTATTCCGGAAGGCATGGTGATTGGTGAAAACGCA1200                          AspArgAlaCysValIleProGluGlyMetValIleGlyGluAsnAla                              385390395400                                                                  GAGGAAGATGCACGTCGTTTCTATCGTTCAGAAGAAGGCATCGTGCTG1248                          GluGluAspAlaArgArgPheTyrArgSerGluGluGlyIleValLeu                              405410415                                                                     GTAACGCGCGAAATGCTACGGAAGTTAGGGCATAAACAGGAGCGATAA1296                          ValThrArgGluMetLeuArgLysLeuGlyHisLysGlnGluArg                                 420425430                                                                     (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 431 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetValSerLeuGluLysAsnAspHisLeuMetLeuAlaArgGlnLeu                              151015                                                                        ProLeuLysSerValAlaLeuIleLeuAlaGlyGlyArgGlyThrArg                              202530                                                                        LeuLysAspLeuThrAsnLysArgAlaLysProAlaValHisPheGly                              354045                                                                        GlyLysPheArgIleIleAspPheAlaLeuSerAsnCysIleAsnSer                              505560                                                                        GlyIleArgArgMetGlyValIleThrGlnTyrGlnSerHisThrLeu                              65707580                                                                      ValGlnHisIleGlnArgGlyTrpSerPhePheAsnGluGluMetAsn                              859095                                                                        GluPheValAspLeuLeuProAlaGlnGlnArgMetLysGlyGluAsn                              100105110                                                                     TrpTyrArgGlyThrAlaAspAlaValThrGlnAsnLeuAspIleIle                              115120125                                                                     ArgArgTyrLysAlaGluTyrValValIleLeuAlaGlyAspHisIle                              130135140                                                                     TyrLysGlnAspTyrSerArgMetLeuIleAspHisValGluLysGly                              145150155160                                                                  ValArgCysThrValValCysMetProValProIleGluGluAlaSer                              165170175                                                                     AlaPheGlyValMetAlaValAspGluAsnAspLysThrIleGluPhe                              180185190                                                                     ValGluLysProAlaAsnProProSerMetProAsnAspProSerLys                              195200205                                                                     SerLeuAlaSerMetGlyIleTyrValPheAspAlaAspTyrLeuTyr                              210215220                                                                     GluLeuLeuGluGluAspAspArgAspGluAsnSerSerHisAspPhe                              225230235240                                                                  GlyLysAspLeuIleProLysIleThrGluAlaGlyLeuAlaTyrAla                              245250255                                                                     HisProPheProLeuSerCysValGlnSerAspProAspAlaGluPro                              260265270                                                                     TyrTrpArgAspValGlyThrLeuGluAlaTyrTrpLysAlaAsnLeu                              275280285                                                                     AspLeuAlaSerValValProLysLeuAspMetTyrAspArgAsnTrp                              290295300                                                                     ProIleArgThrTyrAsnGluSerLeuProProAlaLysPheValGln                              305310315320                                                                  AspArgSerGlySerHisGlyMetThrLeuAsnSerLeuValSerGly                              325330335                                                                     GlyCysValIleSerGlySerValValValGlnSerValLeuPheSer                              340345350                                                                     ArgValArgValAsnSerPheCysAsnIleAspSerAlaValLeuLeu                              355360365                                                                     ProGluValTrpValGlyArgSerCysArgLeuArgArgCysValIle                              370375380                                                                     AspArgAlaCysValIleProGluGlyMetValIleGlyGluAsnAla                              385390395400                                                                  GluGluAspAlaArgArgPheTyrArgSerGluGluGlyIleValLeu                              405410415                                                                     ValThrArgGluMetLeuArgLysLeuGlyHisLysGlnGluArg                                 420425430                                                                     (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1296 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..1296                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       ATGGTTAGTTTAGAGAAGAACGATCACTTAATGTTGGCGCGCCAGCTG48                            MetValSerLeuGluLysAsnAspHisLeuMetLeuAlaArgGlnLeu                              151015                                                                        CCATTGAAATCTGTTGCCCTGATACTGGCGGGAGGACGTGGTACCCGC96                            ProLeuLysSerValAlaLeuIleLeuAlaGlyGlyArgGlyThrArg                              202530                                                                        CTGAAGGATTTAACCAATAAGCGAGCAAAACCGGCCGTACACTTCGGC144                           LeuLysAspLeuThrAsnLysArgAlaLysProAlaValHisPheGly                              354045                                                                        GGTAAGTTCCGCATTATCGACTTTGCGCTGTCTAACTGCATCAACTCC192                           GlyLysPheArgIleIleAspPheAlaLeuSerAsnCysIleAsnSer                              505560                                                                        GGGATCCGTCGTATGGGCGTGATCACCCAGTACCAGTCCCACACTCTG240                           GlyIleArgArgMetGlyValIleThrGlnTyrGlnSerHisThrLeu                              65707580                                                                      GTGCAGCACATTCAGCGCGGCTGGTCATTCTTCAATGAAGAAATGAAC288                           ValGlnHisIleGlnArgGlyTrpSerPhePheAsnGluGluMetAsn                              859095                                                                        GAGTTTGTCGATCTGCTGCCAGCACAGCAGAGAATGAAAGGGGAAAAC336                           GluPheValAspLeuLeuProAlaGlnGlnArgMetLysGlyGluAsn                              100105110                                                                     TGGTATCGCGGCACCGCAGATGCGGTCACCCAAAACCTCGACATTATC384                           TrpTyrArgGlyThrAlaAspAlaValThrGlnAsnLeuAspIleIle                              115120125                                                                     CGTCGTTATAAAGCGGAATACGTGGTGATCCTGGCGGGCGACCATATC432                           ArgArgTyrLysAlaGluTyrValValIleLeuAlaGlyAspHisIle                              130135140                                                                     TACAAGCAAGACTACTCGCGTATGCTTATCGATCACGTCGAAAAAGGT480                           TyrLysGlnAspTyrSerArgMetLeuIleAspHisValGluLysGly                              145150155160                                                                  GTACGTTGTACCGTTGTTTGTATGCCAGTACCGATTGAAGAAGCCTCC528                           ValArgCysThrValValCysMetProValProIleGluGluAlaSer                              165170175                                                                     GCATTTGGCGTTATGGCGGTTGATGAGAACGATAAAACTATCGAATTC576                           AlaPheGlyValMetAlaValAspGluAsnAspLysThrIleGluPhe                              180185190                                                                     GTGGAAAAACCTGCTAACCCGCCGTCAATGCCGAACGATCCGAGCAAA624                           ValGluLysProAlaAsnProProSerMetProAsnAspProSerLys                              195200205                                                                     TCTCTGGCGAGTATGGGTATCTACGTCTTTGACGCCGACTATCTGTAT672                           SerLeuAlaSerMetGlyIleTyrValPheAspAlaAspTyrLeuTyr                              210215220                                                                     GAACTGCTGGAAGAAGACGATCGCGATGAGAACTCCAGCCACGACTTT720                           GluLeuLeuGluGluAspAspArgAspGluAsnSerSerHisAspPhe                              225230235240                                                                  GGCAAAGATTTGATTCCCAAGATCACCGAAGCCGGTCTGGCCTATGCG768                           GlyLysAspLeuIleProLysIleThrGluAlaGlyLeuAlaTyrAla                              245250255                                                                     CACCCGTTCCCGCTCTCTTGCGTACAATCCGACCCGGATGCCGAGCCG816                           HisProPheProLeuSerCysValGlnSerAspProAspAlaGluPro                              260265270                                                                     TACTGGCGCGATGTGGGTACGCTGGAAGCTTACTGGAAAGCGAACCTC864                           TyrTrpArgAspValGlyThrLeuGluAlaTyrTrpLysAlaAsnLeu                              275280285                                                                     GATCTGGCCTCTGTGGTGCCGGAACTGGATATGTACGATCGCAATTGG912                           AspLeuAlaSerValValProGluLeuAspMetTyrAspArgAsnTrp                              290295300                                                                     CCAATTCGCACCTACAATGAATCATTACCGCCAGCGAAATTCGTGCAG960                           ProIleArgThrTyrAsnGluSerLeuProProAlaLysPheValGln                              305310315320                                                                  GATCGCTCCGGTAGCCACGGGATGACCCTTAACTCACTGGTTTCCGAC1008                          AspArgSerGlySerHisGlyMetThrLeuAsnSerLeuValSerAsp                              325330335                                                                     GGTTGTGTGATCTCCGGTTCGGTGGTGGTGCAGTCCGTTCTGTTCTCG1056                          GlyCysValIleSerGlySerValValValGlnSerValLeuPheSer                              340345350                                                                     CGCGTTCGCGTGAATTCATTCTGCAACATTGATTCCGCCGTATTGTTA1104                          ArgValArgValAsnSerPheCysAsnIleAspSerAlaValLeuLeu                              355360365                                                                     CCGGAAGTATGGGTAGGTCGCTCGTGCCGTCTGCGCCGCTGCGTCATC1152                          ProGluValTrpValGlyArgSerCysArgLeuArgArgCysValIle                              370375380                                                                     GATCGTGCTTGTGTTATTCCGGAAGGCATGGTGATTGGTGAAAACGCA1200                          AspArgAlaCysValIleProGluGlyMetValIleGlyGluAsnAla                              385390395400                                                                  GAGGAAGATGCACGTCGTTTCTATCGTTCAGAAGAAGGCATCGTGCTG1248                          GluGluAspAlaArgArgPheTyrArgSerGluGluGlyIleValLeu                              405410415                                                                     GTAACGCGCGAAATGCTACGGAAGTTAGGGCATAAACAGGAGCGATAA1296                          ValThrArgGluMetLeuArgLysLeuGlyHisLysGlnGluArg                                 420425430                                                                     (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 431 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       MetValSerLeuGluLysAsnAspHisLeuMetLeuAlaArgGlnLeu                              151015                                                                        ProLeuLysSerValAlaLeuIleLeuAlaGlyGlyArgGlyThrArg                              202530                                                                        LeuLysAspLeuThrAsnLysArgAlaLysProAlaValHisPheGly                              354045                                                                        GlyLysPheArgIleIleAspPheAlaLeuSerAsnCysIleAsnSer                              505560                                                                        GlyIleArgArgMetGlyValIleThrGlnTyrGlnSerHisThrLeu                              65707580                                                                      ValGlnHisIleGlnArgGlyTrpSerPhePheAsnGluGluMetAsn                              859095                                                                        GluPheValAspLeuLeuProAlaGlnGlnArgMetLysGlyGluAsn                              100105110                                                                     TrpTyrArgGlyThrAlaAspAlaValThrGlnAsnLeuAspIleIle                              115120125                                                                     ArgArgTyrLysAlaGluTyrValValIleLeuAlaGlyAspHisIle                              130135140                                                                     TyrLysGlnAspTyrSerArgMetLeuIleAspHisValGluLysGly                              145150155160                                                                  ValArgCysThrValValCysMetProValProIleGluGluAlaSer                              165170175                                                                     AlaPheGlyValMetAlaValAspGluAsnAspLysThrIleGluPhe                              180185190                                                                     ValGluLysProAlaAsnProProSerMetProAsnAspProSerLys                              195200205                                                                     SerLeuAlaSerMetGlyIleTyrValPheAspAlaAspTyrLeuTyr                              210215220                                                                     GluLeuLeuGluGluAspAspArgAspGluAsnSerSerHisAspPhe                              225230235240                                                                  GlyLysAspLeuIleProLysIleThrGluAlaGlyLeuAlaTyrAla                              245250255                                                                     HisProPheProLeuSerCysValGlnSerAspProAspAlaGluPro                              260265270                                                                     TyrTrpArgAspValGlyThrLeuGluAlaTyrTrpLysAlaAsnLeu                              275280285                                                                     AspLeuAlaSerValValProGluLeuAspMetTyrAspArgAsnTrp                              290295300                                                                     ProIleArgThrTyrAsnGluSerLeuProProAlaLysPheValGln                              305310315320                                                                  AspArgSerGlySerHisGlyMetThrLeuAsnSerLeuValSerAsp                              325330335                                                                     GlyCysValIleSerGlySerValValValGlnSerValLeuPheSer                              340345350                                                                     ArgValArgValAsnSerPheCysAsnIleAspSerAlaValLeuLeu                              355360365                                                                     ProGluValTrpValGlyArgSerCysArgLeuArgArgCysValIle                              370375380                                                                     AspArgAlaCysValIleProGluGlyMetValIleGlyGluAsnAla                              385390395400                                                                  GluGluAspAlaArgArgPheTyrArgSerGluGluGlyIleValLeu                              405410415                                                                     ValThrArgGluMetLeuArgLysLeuGlyHisLysGlnGluArg                                 420425430                                                                     (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 355 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 88..354                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       AAGCTTGTTCTCATTGTTGTTATCATTATATATAGATGACCAAAGCACTAGACCAAACCT60                CAGTCACACAAAGAGTAAAGAAGAACAATGGCTTCCTCTATGCTCTCTTCC111                        MetAlaSerSerMetLeuSerSer                                                      15                                                                            GCTACTATGGTTGCCTCTCCGGCTCAGGCCACTATGGTCGCTCCTTTC159                           AlaThrMetValAlaSerProAlaGlnAlaThrMetValAlaProPhe                              101520                                                                        AACGGACTTAAGTCCTCCGCTGCCTTCCCAGCCACCCGCAAGGCTAAC207                           AsnGlyLeuLysSerSerAlaAlaPheProAlaThrArgLysAlaAsn                              25303540                                                                      AACGACATTACTTCCATCACAAGCAACGGCGGAAGAGTTAACTGCATG255                           AsnAspIleThrSerIleThrSerAsnGlyGlyArgValAsnCysMet                              455055                                                                        CAGGTGTGGCCTCCGATTGGAAAGAAGAAGTTTGAGACTCTCTCTTAC303                           GlnValTrpProProIleGlyLysLysLysPheGluThrLeuSerTyr                              606570                                                                        CTTCCTGACCTTACCGATTCCGGTGGTCGCGTCAACTGCATGCAGGCC351                           LeuProAspLeuThrAspSerGlyGlyArgValAsnCysMetGlnAla                              758085                                                                        ATGG355                                                                       Met                                                                           (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 89 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       MetAlaSerSerMetLeuSerSerAlaThrMetValAlaSerProAla                              151015                                                                        GlnAlaThrMetValAlaProPheAsnGlyLeuLysSerSerAlaAla                              202530                                                                        PheProAlaThrArgLysAlaAsnAsnAspIleThrSerIleThrSer                              354045                                                                        AsnGlyGlyArgValAsnCysMetGlnValTrpProProIleGlyLys                              505560                                                                        LysLysPheGluThrLeuSerTyrLeuProAspLeuThrAspSerGly                              65707580                                                                      GlyArgValAsnCysMetGlnAlaMet                                                   85                                                                            (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1575 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 3..1565                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       CCATGGCGGCTTCCATTGGAGCCTTAAAATCTTCACCTTCTTCTAAC47                             MetAlaAlaSerIleGlyAlaLeuLysSerSerProSerSerAsn                                 151015                                                                        AATTGCATCAATGAGAGAAGAAATGATTCTACACGTGCTGTATCCAGC95                            AsnCysIleAsnGluArgArgAsnAspSerThrArgAlaValSerSer                              202530                                                                        AGAAATCTCTCATTTTCGTCTTCTCATCTCGCCGGAGACAAGTTGATG143                           ArgAsnLeuSerPheSerSerSerHisLeuAlaGlyAspLysLeuMet                              354045                                                                        CCTGTATCGTCCTTACGTTCCCAAGGAGTCCGATTCAATGTGAGAAGA191                           ProValSerSerLeuArgSerGlnGlyValArgPheAsnValArgArg                              505560                                                                        AGTCCAATGATTGTGTCGCCAAAGGCTGTTTCTGATTCGCAGAATTCA239                           SerProMetIleValSerProLysAlaValSerAspSerGlnAsnSer                              657075                                                                        CAGACATGTCTAGACCCAGATGCTAGCCGGAGTGTTTTGGGAATTATT287                           GlnThrCysLeuAspProAspAlaSerArgSerValLeuGlyIleIle                              80859095                                                                      CTTGGAGGTGGAGCTGGGACCCGACTTTATCCTCTAACTAAAAAAAGA335                           LeuGlyGlyGlyAlaGlyThrArgLeuTyrProLeuThrLysLysArg                              100105110                                                                     GCAAAGCCAGCTGTTCCACTTGGAGCAAATTATCGTCTGATTGACATT383                           AlaLysProAlaValProLeuGlyAlaAsnTyrArgLeuIleAspIle                              115120125                                                                     CCTGTAAGCAACTGCTTGAACAGTAATATATCCAAGATTTATGTTCTC431                           ProValSerAsnCysLeuAsnSerAsnIleSerLysIleTyrValLeu                              130135140                                                                     ACACAATTCAACTCTGCCTCTCTGAATCGCCACCTTTCACGAGCATAT479                           ThrGlnPheAsnSerAlaSerLeuAsnArgHisLeuSerArgAlaTyr                              145150155                                                                     GCTAGCAACATGGGAGGATACAAAAACGAGGGCTTTGTGGAAGTTCTT527                           AlaSerAsnMetGlyGlyTyrLysAsnGluGlyPheValGluValLeu                              160165170175                                                                  GCTGCTCAACAAAGTCCAGAGAACCCCGATTGGTTCCAGGGCACGGCT575                           AlaAlaGlnGlnSerProGluAsnProAspTrpPheGlnGlyThrAla                              180185190                                                                     GATGCTGTCAGACAATATCTGTGGTTGTTTGAGGAGCATACTGTTCTT623                           AspAlaValArgGlnTyrLeuTrpLeuPheGluGluHisThrValLeu                              195200205                                                                     GAATACCTTATACTTGCTGGAGATCATCTGTATCGAATGGATTATGAA671                           GluTyrLeuIleLeuAlaGlyAspHisLeuTyrArgMetAspTyrGlu                              210215220                                                                     AAGTTTATTCAAGCCCACAGAGAAACAGATGCTGATATTACCGTTGCC719                           LysPheIleGlnAlaHisArgGluThrAspAlaAspIleThrValAla                              225230235                                                                     GCACTGCCAATGGACGAGAAGCGTGCCACTGCATTCGGTCTCATGAAG767                           AlaLeuProMetAspGluLysArgAlaThrAlaPheGlyLeuMetLys                              240245250255                                                                  ATTGACGAAGAAGGACGCATTATTGAATTTGCAGAGAAACCGCAAGGA815                           IleAspGluGluGlyArgIleIleGluPheAlaGluLysProGlnGly                              260265270                                                                     GAGCAATTGCAAGCAATGAAAGTGGATACTACCATTTTAGGTCTTGAT863                           GluGlnLeuGlnAlaMetLysValAspThrThrIleLeuGlyLeuAsp                              275280285                                                                     GACAAGAGAGCTAAAGAAATGCCTTTCATTGCCAGTATGGGTATATAT911                           AspLysArgAlaLysGluMetProPheIleAlaSerMetGlyIleTyr                              290295300                                                                     GTCATTAGCAAAGACGTGATGTTAAACCTACTTCGTGACAAGTTCCCT959                           ValIleSerLysAspValMetLeuAsnLeuLeuArgAspLysPhePro                              305310315                                                                     GGGGCCAATGATTTTGGTAGTGAAGTTATTCCTGGTGCAACTTCACTT1007                          GlyAlaAsnAspPheGlySerGluValIleProGlyAlaThrSerLeu                              320325330335                                                                  GGGATGAGAGTGCAAGCTTATTTATATGATGGGTACTGGGAAGATATT1055                          GlyMetArgValGlnAlaTyrLeuTyrAspGlyTyrTrpGluAspIle                              340345350                                                                     GGTACCATTGAAGCTTTCTACAATGCCAATTTGGGCATTACAAAAAAG1103                          GlyThrIleGluAlaPheTyrAsnAlaAsnLeuGlyIleThrLysLys                              355360365                                                                     CCGGTGCCAGATTTTAGCTTTTACGACCGATCAGCCCCAATCTACACC1151                          ProValProAspPheSerPheTyrAspArgSerAlaProIleTyrThr                              370375380                                                                     CAACCTCGATATCTACCACCATCAAAAATGCTTGATGCTGATGTCACA1199                          GlnProArgTyrLeuProProSerLysMetLeuAspAlaAspValThr                              385390395                                                                     GATAGTGTCATTGGTGAAGGTTGTGTGATCAAGAACTGTAAGATTCAT1247                          AspSerValIleGlyGluGlyCysValIleLysAsnCysLysIleHis                              400405410415                                                                  CATTCCGTGGTTGGACTCAGATCATGCATATCAGAGGGAGCAATTATA1295                          HisSerValValGlyLeuArgSerCysIleSerGluGlyAlaIleIle                              420425430                                                                     GAAGACTCACTTTTGATGGGGGCAGATTACTATGAGACTGATGCTGAC1343                          GluAspSerLeuLeuMetGlyAlaAspTyrTyrGluThrAspAlaAsp                              435440445                                                                     AGGAAGTTGCTGGCTGCAAAGGGCAGTGTCCCAATTGGCATCGGCAAG1391                          ArgLysLeuLeuAlaAlaLysGlySerValProIleGlyIleGlyLys                              450455460                                                                     AATTGTCACATTAAAAGAGCCATTATCGACAAGAATGCCCGTATAGGG1439                          AsnCysHisIleLysArgAlaIleIleAspLysAsnAlaArgIleGly                              465470475                                                                     GACAATGTGAAGATCATTAACAAAGACAACGTTCAAGAAGCGGCTAGG1487                          AspAsnValLysIleIleAsnLysAspAsnValGlnGluAlaAlaArg                              480485490495                                                                  GAAACAGATGGATACTTCATCAAGAGTGGGATTGTCACCGTCATCAAG1535                          GluThrAspGlyTyrPheIleLysSerGlyIleValThrValIleLys                              500505510                                                                     GATGCTTTGATTCCAAGTGGAATCATCATCTGATGAGCTC1575                                  AspAlaLeuIleProSerGlyIleIleIle                                                515520                                                                        (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 521 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       MetAlaAlaSerIleGlyAlaLeuLysSerSerProSerSerAsnAsn                              151015                                                                        CysIleAsnGluArgArgAsnAspSerThrArgAlaValSerSerArg                              202530                                                                        AsnLeuSerPheSerSerSerHisLeuAlaGlyAspLysLeuMetPro                              354045                                                                        ValSerSerLeuArgSerGlnGlyValArgPheAsnValArgArgSer                              505560                                                                        ProMetIleValSerProLysAlaValSerAspSerGlnAsnSerGln                              65707580                                                                      ThrCysLeuAspProAspAlaSerArgSerValLeuGlyIleIleLeu                              859095                                                                        GlyGlyGlyAlaGlyThrArgLeuTyrProLeuThrLysLysArgAla                              100105110                                                                     LysProAlaValProLeuGlyAlaAsnTyrArgLeuIleAspIlePro                              115120125                                                                     ValSerAsnCysLeuAsnSerAsnIleSerLysIleTyrValLeuThr                              130135140                                                                     GlnPheAsnSerAlaSerLeuAsnArgHisLeuSerArgAlaTyrAla                              145150155160                                                                  SerAsnMetGlyGlyTyrLysAsnGluGlyPheValGluValLeuAla                              165170175                                                                     AlaGlnGlnSerProGluAsnProAspTrpPheGlnGlyThrAlaAsp                              180185190                                                                     AlaValArgGlnTyrLeuTrpLeuPheGluGluHisThrValLeuGlu                              195200205                                                                     TyrLeuIleLeuAlaGlyAspHisLeuTyrArgMetAspTyrGluLys                              210215220                                                                     PheIleGlnAlaHisArgGluThrAspAlaAspIleThrValAlaAla                              225230235240                                                                  LeuProMetAspGluLysArgAlaThrAlaPheGlyLeuMetLysIle                              245250255                                                                     AspGluGluGlyArgIleIleGluPheAlaGluLysProGlnGlyGlu                              260265270                                                                     GlnLeuGlnAlaMetLysValAspThrThrIleLeuGlyLeuAspAsp                              275280285                                                                     LysArgAlaLysGluMetProPheIleAlaSerMetGlyIleTyrVal                              290295300                                                                     IleSerLysAspValMetLeuAsnLeuLeuArgAspLysPheProGly                              305310315320                                                                  AlaAsnAspPheGlySerGluValIleProGlyAlaThrSerLeuGly                              325330335                                                                     MetArgValGlnAlaTyrLeuTyrAspGlyTyrTrpGluAspIleGly                              340345350                                                                     ThrIleGluAlaPheTyrAsnAlaAsnLeuGlyIleThrLysLysPro                              355360365                                                                     ValProAspPheSerPheTyrAspArgSerAlaProIleTyrThrGln                              370375380                                                                     ProArgTyrLeuProProSerLysMetLeuAspAlaAspValThrAsp                              385390395400                                                                  SerValIleGlyGluGlyCysValIleLysAsnCysLysIleHisHis                              405410415                                                                     SerValValGlyLeuArgSerCysIleSerGluGlyAlaIleIleGlu                              420425430                                                                     AspSerLeuLeuMetGlyAlaAspTyrTyrGluThrAspAlaAspArg                              435440445                                                                     LysLeuLeuAlaAlaLysGlySerValProIleGlyIleGlyLysAsn                              450455460                                                                     CysHisIleLysArgAlaIleIleAspLysAsnAlaArgIleGlyAsp                              465470475480                                                                  AsnValLysIleIleAsnLysAspAsnValGlnGluAlaAlaArgGlu                              485490495                                                                     ThrAspGlyTyrPheIleLysSerGlyIleValThrValIleLysAsp                              500505510                                                                     AlaLeuIleProSerGlyIleIleIle                                                   515520                                                                        (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1519 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..1410                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       AACAAGATCAAACCTGGGGTTGCTTACTCTGTGATCACTACTGAAAAT48                            AsnLysIleLysProGlyValAlaTyrSerValIleThrThrGluAsn                              151015                                                                        GACACACAGACTGTGTTCGTAGATATGCCACGTCTTGAGAGACGCCGG96                            AspThrGlnThrValPheValAspMetProArgLeuGluArgArgArg                              202530                                                                        GCAAATCCAAAGGATGTGGCTGCAGTCATACTGGGAGGAGGAGAAGGG144                           AlaAsnProLysAspValAlaAlaValIleLeuGlyGlyGlyGluGly                              354045                                                                        ACCAAGTTATTCCCACTTACAAGTAGAACTGCAACCCCTGCTGTTCCG192                           ThrLysLeuPheProLeuThrSerArgThrAlaThrProAlaValPro                              505560                                                                        GTTGGAGGATGCTACAGGCTAATAGACATCCCAATGAGCAACTGTATC240                           ValGlyGlyCysTyrArgLeuIleAspIleProMetSerAsnCysIle                              65707580                                                                      AACAGTGCTATTAACAAGATTTTTGTGCTGACACAGTACAATTCTGCT288                           AsnSerAlaIleAsnLysIlePheValLeuThrGlnTyrAsnSerAla                              859095                                                                        CCCCTGAATCGTCACATTGCTCGAACATATTTTGGCAATGGTGTGAGC336                           ProLeuAsnArgHisIleAlaArgThrTyrPheGlyAsnGlyValSer                              100105110                                                                     TTTGGAGATGGATTTGTCGAGGTACTAGCTGCAACTCAGACACCCGGG384                           PheGlyAspGlyPheValGluValLeuAlaAlaThrGlnThrProGly                              115120125                                                                     GAAGCAGGAAAAAAATGGTTTCAAGGAACAGCAGATGCTGTTAGAAAA432                           GluAlaGlyLysLysTrpPheGlnGlyThrAlaAspAlaValArgLys                              130135140                                                                     TTTATATGGGTTTTTGAGGACGCTAAGAACAAGAATATTGAAAATATC480                           PheIleTrpValPheGluAspAlaLysAsnLysAsnIleGluAsnIle                              145150155160                                                                  GTTGTACTATCTGGGGATCATCTTTATAGGATGGATTATATGGAGTTG528                           ValValLeuSerGlyAspHisLeuTyrArgMetAspTyrMetGluLeu                              165170175                                                                     GTGCAGAACCATATTGACAGGAATGCTGATATTACTCTTTCATGTGCA576                           ValGlnAsnHisIleAspArgAsnAlaAspIleThrLeuSerCysAla                              180185190                                                                     CCAGCTGAGGACAGCCGAGCATCAGATTTTGGGCTGGTCAAGATTGAC624                           ProAlaGluAspSerArgAlaSerAspPheGlyLeuValLysIleAsp                              195200205                                                                     AGCAGAGGCAGAGTAGTCCAGTTTGCTGAAAAACCAAAAGGTTTTGAT672                           SerArgGlyArgValValGlnPheAlaGluLysProLysGlyPheAsp                              210215220                                                                     CTTAAAGCAATGCAAGTAGATACTACTCTTGTTGGATTATCTCCACAA720                           LeuLysAlaMetGlnValAspThrThrLeuValGlyLeuSerProGln                              225230235240                                                                  GATGCGAAGAAATCCCCCTATATTGCTTCAATGGGAGTTTATGTATTC768                           AspAlaLysLysSerProTyrIleAlaSerMetGlyValTyrValPhe                              245250255                                                                     AAGACAGATGTATTGTTGAAGCTCTTGAAATGGAGCTATCCCACTTCT816                           LysThrAspValLeuLeuLysLeuLeuLysTrpSerTyrProThrSer                              260265270                                                                     AATGATTTTGGCTCTGAAATTATACCAGCAGCTATTGACGATTACAAT864                           AsnAspPheGlySerGluIleIleProAlaAlaIleAspAspTyrAsn                              275280285                                                                     GTCCAAGCATACATTTTCAAAGACTATTGGGAAGACATTGGAACAATT912                           ValGlnAlaTyrIlePheLysAspTyrTrpGluAspIleGlyThrIle                              290295300                                                                     AAATCGTTTTATAATGCTAGCTTGGCACTCACACAAGAGTTTCCAGAG960                           LysSerPheTyrAsnAlaSerLeuAlaLeuThrGlnGluPheProGlu                              305310315320                                                                  TTCCAATTTTACGATCCAAAAACACCTTTTTACACATCTCCTAGGTTC1008                          PheGlnPheTyrAspProLysThrProPheTyrThrSerProArgPhe                              325330335                                                                     CTTCCACCAACCAAGATAGACAATTGCAAGATTAAGGATGCCATAATC1056                          LeuProProThrLysIleAspAsnCysLysIleLysAspAlaIleIle                              340345350                                                                     TCTCATGGATGTTTCTTGCGAGATTGTTCTGTGGAACACTCCATAGTG1104                          SerHisGlyCysPheLeuArgAspCysSerValGluHisSerIleVal                              355360365                                                                     GGTGAAAGATCGCGCTTAGATTGTGGTGTTGAACTGAAGGATACTTTC1152                          GlyGluArgSerArgLeuAspCysGlyValGluLeuLysAspThrPhe                              370375380                                                                     ATGATGGGAGCAGACTACTACCAAACAGAATCTGAGATTGCCTCCCTG1200                          MetMetGlyAlaAspTyrTyrGlnThrGluSerGluIleAlaSerLeu                              385390395400                                                                  TTAGCAGAGGGGAAAGTACCGATTGGAATTGGGGAAAATACAAAAATA1248                          LeuAlaGluGlyLysValProIleGlyIleGlyGluAsnThrLysIle                              405410415                                                                     AGGAAATGTATCATTGACAAGAACGCAAAGATAGGAAAGAATGTTTCA1296                          ArgLysCysIleIleAspLysAsnAlaLysIleGlyLysAsnValSer                              420425430                                                                     ATCATAAATAAAGACGGTGTTCAAGAGGCAGACCGACCAGAGGAAGGA1344                          IleIleAsnLysAspGlyValGlnGluAlaAspArgProGluGluGly                              435440445                                                                     TTCTACATACGATCAGGGATAATCATTATATTAGAGAAAGCCACAATT1392                          PheTyrIleArgSerGlyIleIleIleIleLeuGluLysAlaThrIle                              450455460                                                                     AGAGATGGAACAGTCATCTGAACTAGGGAAGCACCTCTTGTTGAACTA1440                          ArgAspGlyThrValIle                                                            465470                                                                        CTGGAGATCCAAATCTCAACTTGAAGAAGGTCAAGGGTGATCCTAGCACGTTCACCAGTT1500              GACTCCCCGAAGGAAGCTT1519                                                       (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 470 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      AsnLysIleLysProGlyValAlaTyrSerValIleThrThrGluAsn                              151015                                                                        AspThrGlnThrValPheValAspMetProArgLeuGluArgArgArg                              202530                                                                        AlaAsnProLysAspValAlaAlaValIleLeuGlyGlyGlyGluGly                              354045                                                                        ThrLysLeuPheProLeuThrSerArgThrAlaThrProAlaValPro                              505560                                                                        ValGlyGlyCysTyrArgLeuIleAspIleProMetSerAsnCysIle                              65707580                                                                      AsnSerAlaIleAsnLysIlePheValLeuThrGlnTyrAsnSerAla                              859095                                                                        ProLeuAsnArgHisIleAlaArgThrTyrPheGlyAsnGlyValSer                              100105110                                                                     PheGlyAspGlyPheValGluValLeuAlaAlaThrGlnThrProGly                              115120125                                                                     GluAlaGlyLysLysTrpPheGlnGlyThrAlaAspAlaValArgLys                              130135140                                                                     PheIleTrpValPheGluAspAlaLysAsnLysAsnIleGluAsnIle                              145150155160                                                                  ValValLeuSerGlyAspHisLeuTyrArgMetAspTyrMetGluLeu                              165170175                                                                     ValGlnAsnHisIleAspArgAsnAlaAspIleThrLeuSerCysAla                              180185190                                                                     ProAlaGluAspSerArgAlaSerAspPheGlyLeuValLysIleAsp                              195200205                                                                     SerArgGlyArgValValGlnPheAlaGluLysProLysGlyPheAsp                              210215220                                                                     LeuLysAlaMetGlnValAspThrThrLeuValGlyLeuSerProGln                              225230235240                                                                  AspAlaLysLysSerProTyrIleAlaSerMetGlyValTyrValPhe                              245250255                                                                     LysThrAspValLeuLeuLysLeuLeuLysTrpSerTyrProThrSer                              260265270                                                                     AsnAspPheGlySerGluIleIleProAlaAlaIleAspAspTyrAsn                              275280285                                                                     ValGlnAlaTyrIlePheLysAspTyrTrpGluAspIleGlyThrIle                              290295300                                                                     LysSerPheTyrAsnAlaSerLeuAlaLeuThrGlnGluPheProGlu                              305310315320                                                                  PheGlnPheTyrAspProLysThrProPheTyrThrSerProArgPhe                              325330335                                                                     LeuProProThrLysIleAspAsnCysLysIleLysAspAlaIleIle                              340345350                                                                     SerHisGlyCysPheLeuArgAspCysSerValGluHisSerIleVal                              355360365                                                                     GlyGluArgSerArgLeuAspCysGlyValGluLeuLysAspThrPhe                              370375380                                                                     MetMetGlyAlaAspTyrTyrGlnThrGluSerGluIleAlaSerLeu                              385390395400                                                                  LeuAlaGluGlyLysValProIleGlyIleGlyGluAsnThrLysIle                              405410415                                                                     ArgLysCysIleIleAspLysAsnAlaLysIleGlyLysAsnValSer                              420425430                                                                     IleIleAsnLysAspGlyValGlnGluAlaAspArgProGluGluGly                              435440445                                                                     PheTyrIleArgSerGlyIleIleIleIleLeuGluLysAlaThrIle                              450455460                                                                     ArgAspGlyThrValIle                                                            465470                                                                        (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      GTTGATAACAAGATCTGTTAACCATGGCGGCTTCC35                                         (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      CCAGTTAAAACGGAGCTCATCAGATGATGATTC33                                           (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      GTGTGAGAACATAAATCTTGGATATGTTAC30                                              (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      GAATTCACAGGGCCATGGCTCTAGACCC28                                                (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 40 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      AAGATCAAACCTGCCATGGCTTACTCTGTGATCACTACTG40                                    (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      GGGAATTCAAGCTTGGATCCCGGGCCCCCCCCCCCCCCC39                                     (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      GGGAATTCAAGCTTGGATCCCGGG24                                                    (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      CCTCTAGACAGTCGATCAGGAGCAGATGTACG32                                            (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      GGAGTTAGCCATGGTTAGTTTAGAG25                                                   (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      GGCCGAGCTCGTCAACGCCGTCTGCGATTTGTGC34                                          (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      GATTTAGGTGACACTATAG19                                                         (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      AGAGAGATCTAGAACAATGGCTTCCTCTATGCTCTCTTCCGC42                                  (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      GGCCGAGCTCTAGATTATCGCTCCTGTTTATGCCCTAAC39                                     (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2196 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      ATCGATTATTGGTTTATCGGGTTTTGATCGTTATCGGTTCGGTTTAACCGTTAAAATTTG60                ACACAAAAATAAAAATTGAAAAGCACTTAGAAACAAGGTGACAAACCTAATAAACCATGC120               ACATGAGTTCACAAGTTACATCTTGCTAAAAAACAAACACTTTTACATTGTAGAATAACC180               AAGTGTCTGGGACAACCAAAAATGAAAGTAGGAAACCAAACTCTAAGTCAAGGACTTTAT240               ATACAAAATGGTATAACTATAATTATTTAATTTACTATTGGGTTATCGGTTAACCCGTTA300               AGAACCGATAACCCGATAACAAAAACAATCAAAATCGTTATCAAAACCGCTAAACTAATA360               ACCCAATACTGATAAACCAATAACTTTTTTTTTATTCGGGTTATCGGTTTCAGTTCGGTT420               TTGAACAATCCTAGTGTCCTAATTATTGTTTTGAGAACCAAGAAAACAAAAACTGACGTC480               GCAAATATTTCAGTAAATACTTGTATATCTCAGTGATAATTGATTTCCAAGATGTATAAT540               TATCATTTACGTAATAATAGATGGTTTCCGAAACTTACGCTTCCCTTTTTTCTTTTGCAG600               TCGTATGGAATAAAGTTGGATATGGAGGCATTCCCGGGCCTTCAGGTGGAAGAGACGGAG660               CTGCTTCACAAGGAGGGGGTTGTTGTACTTGAAAATAGGCATTTATTCCGTTCGCAAACC720               TATCATGTTCCTATGGTTGTTTATTTGTAGTTTGGTGTTCTTAATATCGAGTGTTCTTTA780               GTTTGTTCCTTTTAATGAAAGGATAATATCTCGTGCCAAAAATAAGCAAATTCGGTACAT840               AAAGACATTTTTTTTCTTTCGTGGATTTTCTGTTTATGGAGTTGTCAAATGTGGAATTTA900               TTTCATAGCATGTGGAGTTTCCTCCTCTCCTTTTTCATGTGCCCTTGGGCCTTGCCTGTT960               TCTTGCACCGCAGTGTGCCAGGGCAGTCGGCAGATGGACATAAATGGCACACCGCTCGGC1020              TCGTGGAAAGAGTATGGTCAGTTTCATTGATAAGTATTTACTCGTATTCGGCGTATACAT1080              CAAGTTAATAGAAAGTAAACACATATGATATCATACATCCATTAGTTAAGTATAAATGCC1140              AACTTTTTACTTGAATCGCTGAATAAATTTACTTACGATTAATATTTAGTTGTGTGTTCA1200              AACATATCATGCATTATTTGATTAAGAATAAATAAACGATGTGTAATTTGAAAACCAATT1260              AGAAAAGAAGTATGACGGGATTGATGTTCTGTGAAATCACTGGCAAATTGAACGGACGAT1320              GAAATTTGATCGTCATTTAAACATATCAACATGGCTTTAGTCATCATCATTATGTTATAA1380              TTATTTTCTTGAAACTTGATACACCAACTCTCATTGGGAAAGTGACAGCATAATATAAAC1440              TATAATATCAATCTGGCAATTTCGAATTATTCCAAATCTCTTTTGTCATTTCATTTCATC1500              CCCTATGTCTGCCTGCAAGTACCAATTATTTAAATACAAAAATCTTGATTAAACAATTCA1560              TTTTCTCACTAATAATCACATTTAATAATAAACGGTTCATACACGTGCGTCACCTTTTTT1620              TCGATTTTCTCTCAAGCGCATGTGATCATATCTAACTCTTGTGCAAACAAGTGAAATGAC1680              GTCCATTAATAAATAATCTTTTGAATACCTGTTCATTTTAATTTATTTGGATTTGCTAAG1740              GATTTTTTTTAGTTTTTGAGATTTTTTATAATTTTAAATTAAAAAAAATAAGTTAAATAT1800              ATCGAAAATGTCTTTTAATCTTATTTTTGAAAAAGATAATTAGCTCAAACAAATTAAAAT1860              TGGTAACTATTTTTCGGAAAAATAATGATTCTTATTGTACATTCTTTTTCATCGATTAGA1920              TATTTTTTTTAAGCTCAAGTACAAAAGTCATATTTCAATCCCCAAAATAGCCTCAATCAC1980              AAGAAATGCTTAAATCCCCAAAATACCCTCAATCACAAAAAGTGTACCAATCATAACTAT2040              GGTCCTCTGTAAATTCCAACAAAATCAAGTCTATAAAGTTACCCTTGATATCAGTACTAT2100              AAAACCAAAAATCTCAGCTGTAATTCAAGTGCAATCACACTCTACCACACACTCTCTAGT2160              AGAGAAATCAGTTGATAACAAGCTTTGTTAACAATG2196                                      (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 591 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      AAGCTTGATATCGAATTCCTGCAGCCCGGGGGATCTCCTTAAAACTTTTTCTGAATTACT60                TTTCAAGATTCTTGATTCTGCACCACTAGCAATTTCCATTTTTCTTTCAGTGATTTTGGT120               TACTTATTTGACATTCTTGTTTTCAAGATCCAACATCATCACTTTCCAGGTTCAAAATCT180               TGTTTTTTTTCTTTTTTCTTTTAATGCTCTATATTGTGGAAGTCCACAGGTGAATTTTTA240               CGATATGGGTTTACCACTTAGCTTTCTTGTAATATTTTATCAATTTTAGAAAATATATGT300               GTGAAATACCTAATTTTACGTAGAGATCATGGGTTCATATGCGTAAAGATTCATGTTTTT360               GTGGTAATGCTATGAGGTATTAGTACTGAGCATATAGCTAGCTTGGGTTTTGGGTTTACC420               GACCAAAAAAAAAAATTAGTGATATTTTCTTTATGTAAATTATACTTTTCTTGGTTGCTA480               AAAGATAACATATACTTTATTGAGATTTGAATAAATCTATTTGATTTAGATCCATTGATA540               AATCTTAATCTTATGGGATTACTGATTTGTTGATTGGCTGCAGAAGGATCC591                        (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1705 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      AAGCTTGGGTACCGGGCCCCCCCTGGAGGTCGAGTGCCATCACATCCAGGGTGTAGGCTC60                GGGGCGTGACAAACTTGGTATCTGAGCTCAGAGTTCAAGAGTCTAAGGTGTCTATAAAGT120               CTGTGCCTTTAGAGTCCTAGTTATCGGTGTGAAGCGCGCCACATCTATAACCAGGAGGCT180               GCGACATTTAAGAATTATCATACTTCTTTCATACTCTTTTCGTGCAATAGAGTTCAACTC240               CATAAAGTCTCTTTATAATTCATGTTTACGCATATCTTTGAGATCATGCCTCCATGTAGA300               GTTGTCTGAGGTCGTCCTGCTAGAAGAAATATTGATCCTCAGGATCAAGGGGTACCCAAT360               GCACCAGAAGTGCGACCCCAAGGAAAGGTCACTAATGTTGAGTTCCAGGATGTTATACGG420               ATATTGAGTGAAGTTGTGACCAACCAAGCTGGACAACAAAGAGGGAATCAACAAGATGTG480               GTTGATACATCCAGAATCCGTGAGTTCTTAAGGATGAATCCTTCAGACTTCACCAATTCA540               AGAGTCACTGAGGATCTGGAAAACTTTGTGGAAGAGTTGTAGAAGGTTTTTGAGGTTATG600               CATGTTGTTGATGCTGAGCGAGTGGAACTAACTGCATACCAACTGAATGGTGTTGCTAGA660               GTATGGTACGACCAATAGAAAAAGAGTAGAGTTGAGGGTGCACAAATTGTGAGTTGGGCA720               GTGTTTGAAGAGGCCTTCATGGGGCATTTCTTTTCCCATGAACTATATGGCAAAGGTAAG780               AGAATTTCCTCACTCTTAAGCAGGAATCCATGAGTGTGCATAAGTATAGCCTCAAGTTCA840               CTCAACTGTCGCCTATGCTCCAGAGATGGCTGTTGATATGAGGAGCAGGATGGGCTTGTT900               TGTGTTTGGGTTGTCTCATCTGTCAATCAAAGAAGGTAAGGTTGTGATGTGGATAAAGGA960               CATGGACATCGAAAGGGTAATGATCCTTGTGCAACAGGTTGAGGAAGATAAGTTGAGGGA1020              TAGAGAAGAGTTCTGAAACAAGAGGGCTAAGAACACATGAAATGAGTACGTAAGCAGAAG1080              AGTAATGCAAATCGGTTATCTTTTCAATGAAAGCCAAATAAACCTGCTTGATTGTTTGCA1140              AGTGCAACCTGTACCAACGAACAAAGGTGAGTTCAAGAATCAGAATTCTTAGAAATTCAG1200              AGCTAGACCTGCACAATCTCAAGGTAGTGTGGCACAAGGATGTAATGGGACTCCTGCATG1260              TGTTAAGTACGGTAGGAACCACCCAGGAGCGTGTCATGATGGCTCTGCTGGTTGCTTCAA1320              GTGTGGTCAGAATGGTCACTTCATGAGAGAGTGCCTAAAGAANAGGCAAGGTAATAGCAA1380              TGGGGGCAATATATCACAATCTTCTTCAGTGGCTCCACNAGATAGAGCTGCACCTTGAGG1440              ATCATGGGTTCATATGCGTAAAGATTCATGTTTTGTGGTAATGCTATGAGGTATTAGTAC1500              TGAGCATATAGCTAGCTTGGGTTTTGGGTTTACCGACCATTTTTTTTAATTAGTGATATT1560              TTCTTTATGTATTTTATACTTTTCTTGGTTGCTTAAAGATTACATATACTTTATTGAGAT1620              TTGAATAAATCTATTTGATTTAGATCCATTGATAAATCTTAATCTTATGGGATTACTGAT1680              TTGTTGATTGGCTGCAGAAGGATCC1705                                                 __________________________________________________________________________

We claim:
 1. A method of reducing the level of sugars within potatotubers stored at reduced temperatures comprising providing an increasedlevel of ADPglucose pyrophosphorylase enzyme activity during storage atreduced temperatures by transforming potato plants with a recombinant,double-stranded DNA molecule comprising (a) a promoter which functionsin potato tubers to cause the production of an RNA sequence in tubers,(b) a structural DNA sequence that causes the production of an RNAsequence which encodes a fusion polypeptide comprising an amino-terminalplastid transit peptide and a foreign ADPglucose pyrophosphorylaseenzyme, and (c) a 3' nontranslated DNA sequence which functions in plantcells to cause transcriptional termination and the addition ofpolyadenylated nucleotides to the 3' end of the RNA sequence; obtainingtubers from such transformed potato plants; and storing said tubers atreduced temperatures without formation of unacceptably high sugarlevels.
 2. The method of claim 1 wherein said ADPGPP enzyme is the E.coli glgC enzyme.
 3. The method of claim 1 wherein said ADPGPP enzyme isa mutant E. coli enzyme.
 4. The method of claim 3 wherein said ADPGPPenzyme has the sequence shown in SEQ ID NO:4.
 5. A method of prolongingdormancy of stored potato tubers comprising providing an increased levelofADPglucose pyrophosphorylase enzyme activity within the tuber duringstorage by transforming potato plants with a recombinant,double-stranded DNA molecule comprising (a) a promoter which functionsin potatoes to cause the production of an RNA sequence in tubers, (b) astructural DNA sequence that causes the production of an RNA sequencewhich encodes a fusion polypeptide comprising an amino-terminal plastidtransit peptide and a foreign ADPglucose pyrophosphorylase enzyme, and(c) a 3' nontranslated DNA sequence which functions in plant cells tocause transcriptional termination and the addition ofpolyadenylatednucleotides to the 3' end of the RNA sequence; and obtaining tubers fromsuch transformed potato plants which exhibit inhibited sprouting duringstorage.
 6. The method of claim 5 wherein said storage is at reducedtemperatures.
 7. The method of claim 6 wherein said ADPGPP enzyme is theE. coli glgC16 enzyme.
 8. The method of claim 6 wherein said ADPGPPenzyme has the sequence shown in SEQ ID NO:4.